Probes, methods and kits for detection and typing of Helicobacter pylori nucleic acids in biological samples

ABSTRACT

The present invention relates to a method for the detection and/or typing of  Helicobacter pylori  ( H. pylori ) strains present in a sample comprising the steps of: (i) if need be releasing, isolating or concentrating the polynucleic acids in the sample, (ii) amplifying the polynucleic acids of relevant target regions of the vacA gene and possibly other virulence determinant genes (VDG), with suitable primer pairs, said primers being generally applicable on different  H. pylori  strains, allowing to amplify said relevant target regions of the VDG preferentially in compatible amplification conditions; (iii) hybridizing the polynucleic acids obtained in (i) or (ii) with a set of at least two VDG-derived probes, under appropriate hybridization and wash conditions, and with at least one of said probes hybridizing to a conserved region of a VDG of  H. pylori , and with at least one of said probes hybridizing to a variable region of vacA; (iv) detecting the hybrids formed in step (iii), (v) detecting and/or typing  H. pylori  strains present in a sample from the differential hybridization signals obtained in step (iv), with said typing being the allele-specific detection of a strain according to the VDG alleles present in that particular  H. pylori  strain, and the said virulence determinant genes being the genetic elements involved in enabling, determining, and marking of the infectivity and/or pathogenicity of said  H. pylori  strain. The present invention also relates to probes and primers for doing the same as well as  Helicobacter pylori  detecting/typing kits. The present invention also discloses novel sequences of VDG, which can be used for designing the above-mentioned primers and probes.

[0001] This invention relates to the field of the detection and typingof the human pathogen Helicobacter pylori, abbreviated as H.pyloribelow.

[0002] This invention relates to probes, primers, methods, and kitscomprising the same for the detection and typing of nucleic acids ofH.pylori in biological samples.

[0003]H.pylori is the causative agent of chronic superficial gastritisin humans and infection with this organism is a significant risk factorfor the development of peptic ulcer disease and gastric cancer. (Blaseret al. 1992; Hentschel et al. 1993; Parsonnet et al., 1991)

[0004] The outcome of an infection with H.pylori is rather diverse,probably reflecting the large diversity within the species at thegenetic level (Foxall et al., 1992: Akopyanz et al., 1992). However,most phenotypic characteristics are well conserved. As individuals canbe infected with various strains, it will however be important toidentify particular characteristics of different H.pylori strains thatprecisely determine risk among these strains.

[0005] Among the respective virulence determinants of H.pylori, twoimportant genetic elements have been identified recently the vacuolatingtoxin gene (vacA gene) and the cytotoxin associated gene (cagA gene)(Leunk et al 1988: Cover and Blaser, 1992, 1995; Cover et al. 1992,1994, Tummuru et al., 1993; Covacci et al. 1993).

[0006] The H.pylori vacuolating toxin induces cytoplasmic vacuolation ina large number of mammalian cell lines in vitro (Leunk et al. 1988), andproduces epithelial cell damage and mucosal ulceration whenadministrated intragastrically to mice(Telford et al., 1993). The vacAgene encodes a 1287-1296 amino acid precursor which is processed (N- andC-terminally) to a 87-Kda secreted protein (Cover and Blaser, 1992;Cover et al., 1994; Telford et al. 1994; Schmitt and Haas, 1994; Phadniset al., 1994). Although only 50% of the H.pylori strains inducevacuolation, nearly all strains hybridize to vacA probes (Cover et al.1994: Telford et al., 1994; Schmitt and Haas 1994; Phadnis et al. 1994).Very recently, Atherton et al. (1995) gave evidence for a mosaicorganisation of the vacA gene, which indicated that specific vacAgenotypes of H.pylori swains are associated with the level of cytotoxinactivity, in vitro as well as with the clinical consequences.

[0007] It was shown that three different classes of vacA signalsequences (s1a, s2b and S2) are present and two different classes ofmiddle-region alleles (m2 and m2). All possible combinations of thesevacA regions have been isolated, with the exception of s2/m1. Theproduction of cytotoxin activity was strongly linked to the presence ofvacA alleles containing the s1-type signal peptide. None of the strainscontaining s2-type vacA alleles produced detectable cytotoxin activity.Also, a significant correlation between the occurrence of pepticulceration and the presence of s1-type vacA alleles could bedemonstrated

[0008] A second putative virulence determinant is the high molecularweight protein encoded by the cytotoxin-associated gene, cagA (Tummuruet al., 1993; Covacci et al., 1993). About 60% of the H pylori strainspossess the cagA gene and nearly all of them express the cagA geneproduct Production of the vacuolating cytotoxin in vitro and thepresence of cagA are closely associated characteristics, although bothgenes are not tightly genetically linked (Turn et al., 1993; Covacci etal, 1993).

[0009] Based on immumoblot studies, it has been demonstrated thatpersons infected with cagA(+)-strains have higher degrees of gastricinflammation and epithelial cell damage in comparison to infections withcagA(+)-strains Also, an inhanced expression of a number of cytokineshas been found with respect to infection with cagA(+)-strains incomparison to cagA(−)-strains (Huang et al.,1995). As both the intensityof the inflammmation and the degree of epithelial damage may bedetermining the pathogenesis of gastric cancer, the examination of thepresence or abscence of the cagA gene upon H.pylori infection isimportant.

[0010] In this invention, it is disclosed for the first time that themethods described by Atherton et al., 1995 are not suitable to typeH.pylori strains present in a number of clinical samples obtained frompatients of the Netherlands and Portugal (see example 1). Moreover, thetyping method described by these authors involves the resolution ofgene-amplification products by agarose gel electrophoresis, a tediousand not highly reliable technique when applied on large number ofsamples.

[0011] Thus, with respect to the nessecity to evaluate large populationsto provide statistically relevant data concerning the linkage between atype of H.pylori strains and any pathogenic phenotype and in view of theneed for a rapid, simple and highly reliable typing method in order todetermine the applicable eradication strategy at the clinical stage, theabove method described by Atherton et al, 1995 is less appropriate.

[0012] It is an aim of this present invention to provide a rapid,sensitive and reliable method to detect and type H. pylori strains inbiological samples.

[0013] More particularly, it is an aim of the present invention toprovide a rapid, sensitive and reliable method to detect and/or typeH.pylori strains in biological samples, associated with the developmentof chronic active gastritis and/or gastric and duodenal ulcers, and/orgastric adenocarcinomas and/or mucosa-associated lymphoid tissuelymphomas, and/or to determine the applicable eradication therapy.

[0014] It is an aim of the present invention to provide a rapid,sensitive and reliable method to detect and type H.pylori strainspresent in biological samples, directly coupled to the detection and/orthe typing of the alleles of the virulence determinant genes present,including at least the vacA gene.

[0015] More particularly, it is an aim of the present invention toprovide a rapid, sensitive and reliable method to detect and type H.pylori strains present in a biological sample, directly coupled to thedetection and/or the typing of the vacA and cagA alleles present.

[0016] It is the aim of the present invention to define suitable probesenabling the detection and/or allele-specific typing of H.pylori strainsbased on the alleles of the virulence determinant genes present,including at least one probe derived from vacA More particularly, it isan aim of the present invention to define suitable probes enabling thedetection and/or allele-specific typing of H pylori strains based on thealleles of the vacA and cagA virulence determinant genes present.

[0017] It is moreover an aim of the present invention to combine thesuitable probes enabling detection and/or allele-specific typing ofH.pylori strains based on the alleles of the virulence determinant genespresent, including at least the vacA gene, whereby all said probes canpreferentially be used simultanously in a multiparameter type of assay,more particularly under the same hybridisation and wash-conditions.

[0018] More particularly, it is an aim of the present invention tocombine the suitable probes enabling detection and/or allele-specifictyping of H.pylori strains based on the alleles of the vacA and cagAgenes present, whereby all probes can be preferentially usedsimultanously under the same hybridisation and wash-conditions.

[0019] More particularly, it is an aim of this invention to developsuitable probes of relevant target regions of the VDG, including atleast the vacA gene, said target regions comprising either a variableregion, either a conserved region of the VDG, said probes beingapplicable, if appropriate, in a simultanous hybridisation assay.

[0020] Even more particularly, it is an aim of this invention to developsuitable probes of relevant target regions of the vacA and cagA genes,said target regions comprising a variable region in case of the vacAgene and a conserved region in case of the cagA gene, said probes beingapplicable, if appropriate, in a simultanous hybridisation assay.

[0021] Most particularly, it is an aim of this invention to designsuitable probes comprising the highly variable S- and M-regions in thevacA gene, said S-region being comprised between the nucleotides atposition 1 and 300, and said M-regions being comprised between thenucleotides at the position 1450 and 1650, and a common probe in thecase of the cagA gene comprising preferentially the highly conservedregion between the nucleotide at the position 17 and the nucleotide atthe position 113 of the cagA gene of H.pylori, if appropriate, in asimultanous hybridisation assay.

[0022] It is also an aim of the present invention to select primersenabling the amplification of relevant target regions of alleles of thevirulence determinant gene of interest of H.pylori including at leastthe vacA gene, said amplification being universal for the respectivetarget regions, said target regions comprising either a variable regionor a conserved region of the VDG.

[0023] It is more particularly an aim of the present invention to selectprimers enabling the amplification of the relevant target regions of thealleles of the vacA and cagA virulence determinant genes of theH.pylori, said primers being being generally applicable with H.pyloristrains and allowing the amplification of said relevant target regionsto be used in compatible amplification conditions said amplificationbeing universal for the respective vacA and cagA alleles present.

[0024] Most particularly, it is an aim of the present invention toselect primers enabling the amplification of the highly variable S- andM-regions in the vacA gene, said S-region being comprised between thenucleotide at position 1 and 300, said M-region being comprised betweenthe nucleotides at the position 1450 and 1650, and the highly conservedregion between the nucleotide at the position 1 and the nucleotide atthe position 250 of the open reading frame of the cagA gene of H.pylori,by preference in a single amplification reaction. It is also an aim ofthe present invention to provide kits for the detection and/or typing ofH.pylori subs.

[0025] More particularly, it is an aim of this invention to provide akit for the detection and/or typing of H.pylori strains directly coupledto the detection and/or the typing of the alleles of the virulencedeterminant genes present, including at least the vacA gene. Even moreparticularly, it is an aim of this invention to provide a kit for thedetection and/or typing of H.pylori strains based on the detectionand/or typing of the alleles of the vacA and cagA genes present.

[0026] Most preferentially, it is an aim of this invention to provide akit for the detection and/or typing Of H.pylori stains based on thedetection and/or typing of the highly variable S- and M-regions in thevacA gene and the highly conserved region between the nucleotide at theposition 1 and the nucleotide at the position 250 of the cagA gene ofH.pylori.

[0027] All the aims of the present invention have been met by thefollowing specific embodiments. The selection of the probes (except forprobes with SEQ ID NO 35 to 39) according to the present invention isbased on the Line Probe Assay (LPA) principle, as exemplified in theExamples section. The LiPA is a reverse hybridization assay usingoligonucleotide probes immobilized as parallel lines on a solid supportstrip (Stuyver et al. 1993; international application WO 94/12670). Thisapproach is particularly advantageous since it is fast and simple toperform. The reverse hybridization format and more particularly the LiPAapproach has many practical advantages as compared to other DNAtechniques or hybridization formats, especially hen the use of acombination of probes is preferable or unavoidable to obtain therelevant information sought. As such, the LiPA is a particularlyappropriate method to detect and or type (micro)-organisms in generaland H.pylori in particular. The probes with SEQ ID NO 35 to 39 aredesigned for use in a DNA Immuno Assay, as shown in example 8. Thisassay is particularly convenient for a rapid detection method.

[0028] It is to be understood, however, that any other type ofhybridization assay or hybridization format using any of the selectedprobes as described further in the invention, is also covered by thepresent invention.

[0029] The reverse hybridization approach implies that the probes areimmobilized to a solid support and that the target DNA is labelled inorder to enable the detection of the hybrids formed. The followingdefinitions serve to illustrate the terms and expressions used in thepresent invention.

[0030] The target material in the samples envisaged in the presentinvention may either be DNA or RNA e.g. genomic DNA or messenger RNA oramplified versions thereof. These molecules are also termed polynucleicacids.

[0031] The relevant target regions will in principle be all polynucleicacid sequences comprising a virulence determinant gene, said virulencedeterminant gene being the genetic element involved in enabling,determining, and marking of the infectivity and/or pathogenecity ofH.pylori, more specifically all polynucleic acid sequences comprisingthe virulence determinant genes vacA and cagA, and even morespecifically any conserved region in the cagA gene, said conservedregion being defined as more being more than 95% identical betweenalleles of different H.pylori strains, and most specifically thevariable S- and M-regions of the vacA gene. In addition to variablesequences, the S-region of the vacA gene also comprises conservedsequences, which may be chosen as target regions for probes fordetection—without typing—of H. pylori according to the presentinvention.

[0032] The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence to be detected.

[0033] The term complementary as used herein means that the sequence ofthe single stranded probe is exactly hybridizing to the sequence of thesingle-stranded target, with the target being defined as the sequencewhere the mutation to be detected is located. Since the currentapplication requires the detection of single basepair mismatches, verystringent conditions for hybridization are required, allowing inprinciple only hybridization of exactly complementary sequences.However, variations are possible in the length of the probes (seebelow), and it should be noted that, since the central part of the probeis essential for its hybridization characteristics, possible deviationsof the probe sequence versus the target sequence may be allowabletowards bead and tail of the probe, when longer probe sequences areused. These variations, which may be conceived from the common knowledgein the art, should however always be evaluated experimentally, in orderto check if they result in equivalent hybridization characteristicscompared to the exactly complementary probes.

[0034] Preferably, the probes are about 5 to 50 nucleotides long, morepreferably from about 10 to 25 nucleotides. The nucleotides as used inthe present invention may be ribonucleotides, deoxyribonucleotides andmodified nucleotides such as inosine or nucleotides containing modifiedgroups which do not essentially alter their hybridisationcharacteristics.

[0035] Probe sequences are represented throughout the specification assingle stranded DNA oligonucleotides from the 5′ to the 3′ end. It isobvious to the man skilled in the art that any of the below-specifiedprobes can be used as such, or in their complementary form, or in theirRNA form (wherein T is replaced by U).

[0036] The probes according to the invention can be prepared by cloningof recombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

[0037] The term “solid support” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead) or a chip. Prior to application to the membrane orfixation it may be convenient to modify the nucleic acid probe in orderto facilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

[0038] The term “labelled” refers to the use of labelled nucleic acids.Labelling may be carried out by the use of labelled nucleotidesincorporated during the polymerase step of the amplification such asillustrated by Saiki et al. (1988) or Bej et al. (1990) or labelledprimers, or by any other method known to the person skilled in the art.The nature of the label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic(biotin, digoxigenin, etc.).

[0039] The team “primer” refers to a single stranded oligonucleotidesequence capable of acting as a point of initiation for synthesis of aprimer extension product which is complementary to the nucleic acidstrand to be copied. The length and the sequence of the primer must besuch that they allow to prime the synthesis of the extension products.Preferably the primer is about 5-50 nucleotides long. Specific lengthand sequence will depend on the complexity of the required DNA or RNAtargets, as well as on the conditions of primer use such as temperatureand ionic strenght.

[0040] The fact that amplification primers do not have to match exactlywith the corresponding template sequence to warrant proper amplificationis amply documented in the literature (Kwok et al., 1990).

[0041] The amplification method used can be either polymerase chainreaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgrenet al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acidsequence-based amplification (NASBA; Guatelli et at, 1990; Compton,1991), transcription-based amplification system (TAS; Kwoh et al, 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of QB replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules known in the art.

[0042] The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al., 1979) Or peptide nucleic acids(Nielsen et al., 1991; Nielsen et al., 1993) or may containintercalating agents (Asseline et al., 1984).

[0043] As for most other variations or modifications introduced into theoriginal DNA sequences of the invention, these variations willnecessitate adaptations with respect to the conditions under which theoligonucleotide should be used to obtain the required specificity andsensitivity. However the eventual results of hybridisation will beessentially the same as those obtained with the unmodifiedoligonucleotides.

[0044] The introduction of these modifications may be advantageous inorder to positively influence characteristics such as hybridizationkinetics, reversibility of the hybrid-formation, biological stability ofthe oligonucleotide molecules, etc.

[0045] The “sample” may be any biological material taken either directlyfrom the infected human being (or animal), or after culturing(enrichment), or collected from any other environment. Biologicalmaterial may be e.g. expectorations of any kind, broncheolavages, blood,skin tissue, biopsies, lymphocyte blood culture material colonies,liquid cultures, soil, faecal samples, urine, surface water, etc.

[0046] The probes of the invention are designed for attaining optimalperformance under the same hybridization conditions so that they can beused in sets for simultaneous hybridization; this highly increases theusefulness of these probes and results in a significant gain in time andlabour. Evidently, when other hybridization conditions would bepreferred, all probes should be adapted accordingly by adding ordeleting a number of nucleotides at their extremities. It should beunderstood that these concommitant adaptations should give rise toessentially the same result, namely that the respective probes stillhybridize specifically with the defined target. Such adaptations mightalso be necessary if the amplified material should be RNA in nature andnot DNA as in the case for the NASBA system

[0047] For designing probes with desired characteristics, the followinguseful guidelines known to the person skilled in the art can be applied.

[0048] Because the extent and specificity of hybridization reactionssuch as those described herein are affected by a number of factors,manipulation of one or more of those factors will determine the exactsensitivity and specificity of a particular probe, whether perfectlycomplementary to its target or not. The importance and effect of variousassay conditions, explained further herein, are known to those skilledin the art.

[0049] First, the stability of the [probe:target] nucleic acid hybridshould be chosen to be compatible with the assay conditions. This may beaccomplished by avoiding long AT-rich sequences, by terminating thehybrids with G:C base pairs. and by designing the probe with anappropriate Tm The beginning and end points of the probe should bechosen so that the length and % GC result in a Tm about 2-10° C. higherthan the temperature at which the final assay will be performed. Thebase composition of the probe is significant because G-C base pairsexhibit greater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

[0050] Conditions such as ionic strenght and incubation temperatureunder which a probe will be used should also be taken into account whendesigning a probe. It is known that hybridization will increase as theionic strenght of the reaction mixture increases, and that the thermalstability of the hybrids will increase with increasing ionic strenght.On the other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the T_(m). In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplexIncubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.It is desirable to have probes which hybridize only under conditions ofhigh stringency. Under high stringency conditions only highlycomplementary nucleic acid hybrids will form; hybrids without asufficient degree of complementarity will not form Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. The degree of stringency is chosen such as to maximize thedifference in stability between the hybrid formed with the target andthe nontarget nucleic acid. Second, probes should be positioned so as tominimize the stability of the [probe:nontarget] nucleic acid hybrid.This may be accomplished by minimizing the length of perfectcomplementarity to non-target organisms, by avoiding GC-rich regions ofhomology to non-target sequences, and by positioning the probe to spanas many destabilizing mismatches as possible. Whether a probe sequenceis useful to detect only a specific type of organism depends largely onthe thermal stability difference between [probe:target] hybrids and[probe:nontarget] hybrids. In designing probes, the differences in theseTm values should be as large as possible (e.g. at least 2° C. andpreferably 5° C.).

[0051] The length of the target nucleic acid sequence and, accordingly,the length of the probe sequence can also be important. In some cases,there may be several sequences from a particular region, varying inlocation and length, which will yield probes with the desiredhybridization characteristics. In other cases, one sequence may besignificantly better than another which differs merely by a single base.While it is possible for nucleic acids that are not perfectlycomplementary to hybridize, the longest stretch of perfectlycomplementary base sequence will normally primarily determine hybridstability. While oligonucleotide probes of different lengths and basecomposition may be used, preferred oligonucleotide probes of thisinvention are between about 5 to 50 (more particularely 10-25) bases inlength and have a sufficient stretch in the sequence which is perfectlycomplementary to the target nucleic acid sequence.

[0052] Third, regions in the target DNA or RNA which are known to formstrong internal structures inhibitory to hybridization are lesspreferred. Likewise, probes with extensive self-complementarity shouldbe avoided. As explained above, hybridization is the association of twosingle strands of complementary nucleic acids to form a hydrogen bondeddouble strand. It is implicit that if one of the two strands is whollyor partially involved in a hybrid that it will be less able toparticipate in formation of a new hybrid. There can be intramolecularand intermolecular hybrids formed within the molecules of one type ofprobe if there is sufficient self complementarity. Such structures canbe avoided through carefull probe design. By designing a probe so that asubstantial portion of the sequence of interest is single stranded, therate and extent of hybridization may be greatly increased. Computerprograms are available to search for this type of interaction. However,in certain instances, it may not be possible to avoid this type ofinteraction.

[0053] The present invention provides in its most general form a methodfor the detection and/or typing of Helicobacter pylori (H.pylori)strains present in a sample comprising the steps of:

[0054] (i) if need be releasing, isolating or concentrating thepolynucleic acids in the sample;

[0055] (ii) amplifying the polynucleic acids of relevant target regionsof the vacA gene and possibly other virulence determinant genes (VDG),with suitable primer pairs, said primers being generally applicable ondifferent H.pylori strains, allowing to amplify said relevant targetregions of the VDG preferentially in compatible amplificationconditions;

[0056] (iii) hybridizing the polynucleic acids obtained in (i) or (ii)with a set of at least two VDG-derived probes, under appropriatehybridization and wash conditions, and with at least one of said probeshybridizing to a conserved region of a VDG of H.pylori, and with atleast one of said probes hybridizing to a variable region of vacA;

[0057] (iv) detecting the hybrids formed in step (iii);

[0058] (v) detecting and/or typing H.pylori strains present in a samplefrom the differential hybridization signals obtained in step (iv).

[0059] Said typing represents the allele-specific detection of a strainaccording to the VDG alleles present in that particular H.pylori strain.Said virulence determinant genes represent the genetic elements involvedin enabling, determining, and marking of the infectivity and/orpathogenicity of said H.pylori strain. Said method is referred to belowas “detection/typing method”.

[0060] The relevant target regions will be derived from polynucleic acidsequences comprising a virulence determinant gene specific of H.pylori,with said relevant target region being either a conserved region in aVDG, or a variable region of a VGD. The relevant target regions of thevirulence determinant genes relate either to any conserved region inknown VDG, allowing detection of the presence of this VDG in theH.pylori strains in a sample, or to any variable region in known VDGallowing allele-specific typing of the H.pylori present in a sample.

[0061] According to a preferred embodiment of the present invention,step (ii) and (iii) are performed using primers and probes meticulouslydesigned such that they show the desired amplification or hybridizationresults, when used, if appropriate under compatible amplification orhybridization and wash conditions.

[0062] More specifically, the present invention provides a method forthe detection and/or typing of H. pylori strains present in a samplewith respect to the development of chronic active gastritis and/orgastric and duodenal ulcers and/or gastric adenocarcinomas and/ormucosa-associated lymphoid tissue lymphomas and/or determiningeradication therapy.

[0063] The cagA gene and the vacA gene are representatives of thevirulence determinant genes of H.pylori. Relevant conserved largestregions of alleles of the cagA gene can be used to detect the presenceof this gene in H.pylori strains present in a sample. In addition,identified variable regions in alleles of the vacA gene can be used totype in an allele-specific way the respective H.pylori strains. Bypreference said conserved target regions of alleles of the cagA geneinclude the region spanning the nucleotide at position 1 to thenucleotide at the position 250 of the open reading frame with saidnumbering being according to Genbank accessions L11741 (HECMAJANT) orX70039 (HPCAI); also, by preference the identified variable regions ofalleles of the vacA gene include the identified S- and M-region of thevacA gene, said S-region being comprised between the nucleotides atposition 1 and 300, said M-region being comprised between thenucleotides at the position 1450 and 1650, with said numbering beingaccording to Genbank accessions U05676 or U29401.

[0064] Standard hybridization and wash conditions are for instance 2×SSC(Sodium Saline Citrate), 0.1% SDS at 50° C. Other solutions (SSPE(Sodium Saline phosphate EDTA), TMACI (Tetramethyl ammonium Chloride),etc) and temperatures can also be used provided that the specificity andsensitivity of the probes is maintained. If need be, slightmodifications of the probes in length or in sequence might have to becarried out in order to maintain the specificity and sensitivityrequired under the given conditions. Suitable primers can for instancebe chosen form a list of primers described below.

[0065] In a more preferential embodiment, the above mentionedpolynucleic acids from step (ü) are hybridized with at least two, three,four, five or more of the above mentioned cagA- or vacA-derived probes,which cover respectively a conserved region of the cagA gene and avariable region of the vacA gene.

[0066] Also, in a more preferential embodiment, the above mentionedpolynucleic acids from step (i) and (ii) are hybridized with at leastone vacA-derived probe directed to at least one identified variableregion of the alleles of the vacA gene, by preference including at leastone of the vacA-derived probes SEQ ID NO 2 to 11 and 28 to 34.

[0067] It should be stressed that all of the above-mentioned probes,including the allele-specific probes, are contained in the sequence ofspecific virulence determinant genes of H.pylori, including moreparticularly the cagA gene or the vacA gene, said probes comprisingeither a conserved region of the cagA gene, or comprising a variableregion of the vacA gene. The probes are preferably designed in such away that they can all be used simultanously, under the samehybridization and wash conditions. Both criteria imply thatpreferentially a single amplification and hybridization step issufficient for the simultanous detection and typing of H.pylori strainspresent in a sample.

[0068] The present invention relates more particularly to a method asdefined above wherein step (ii) consists of amplifying the polynucleicacids of relevant target regions in the vacA and cagA gene with suitablesets of primers, said primers being generally applicable on different H.pylori strains, allowing to amplify said relevant target regions incompatible amplification conditions, with said target region being aconserved region in the case of the cagA alleles and a variable regionin the case of the vacA alleles, and with said sets of primers beingpreferentially chosen from the following list of primers as given inTable 1: cagF (SEQ ID NO 12) cagR (SEQ ID NO 13) VA1XR (SEQ ID N0 14)VA1F (Atherton et al, 1995) M1F (SEQ ID NO 15) M1R (SEQ ID NO 16) HPMGF(SEQ ID NO 17) HPMGR (SEQ ID NO 18) cagSF (SEQ ID NO 19) cagSR (SEQ IDNO 20) cagFN1 (SEQ ID NO 21) cagRN1 (SEQ ID NO 22) VAMSFb (SEQ ID NO 23)VAMSFc (SEQ ID NO 24) VAMSFd (SEQ ID NO 25) VAMSFe (SEQ ID NO 26)

[0069] or sequence variants thereof; with said sequence variantscontaining deletions and/or insertions and/or substitutions of one ormore nucleotides, mainly at their extremities (either 3′ or 5′), and orsubstitutions of non-essential nucleotides, —being nucleotides notessential in discriminating between alleles—, by others (includingmodified nucleotides such as inosine), or with said variants conning ofthe complement of any of the above-mentioned oligonucleotide primers, orwith said variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that the variants canhybridize/amplify specifically with the same specificity as theoligonucleotide primers from which they are derived.

[0070] Primers cagF and cagR are derived from two published sequences ofcagA alleles (Cocacci et al, 1993; Tummuru et al., 1993). The presentinvention provides novel nucleic acid sequences encoding 149-154 aminoacids of the N-terminus of the cagA protein, as disclosed in FIG. 10(see also example 5). Based on these novel sequences, improved primerswere designed for amplification of a relevant target region of the cagAgene. These primers are: cagSF(forward) (SEQ ID NO 19) cagSk(reverse)(SEQ ID NO 20)

[0071] The sequence of these primers is shown in table 1. Study of thealignment of sequences shown in FIG. 10 shows that primers cagSF andcagSR will not hybridize to the polynucleic acids of isolates from EastAsia. Therefore, even more improved primers were designed, that willalso permit amplification of these sequences. These primers are:cagFN1(forward) (SEQ ID NO 21) cagRN1(reverse) (SEQ ID NO 22)

[0072] The sequence of these primers is shown in table 1. Primers cagSFand cagSR can of course be used when amplification of polynucleic acidsof isolates from East Asia is not required. Primers M1F. M1R, HPMGF andHPMGR are based on the sequences of the M-region of the vacA gene, shownin FIGS. 2 and 3, said sequences being provided by the presentinvention. In a second instance, the present invention disclosesadditional sequences for the M-region, as shown in FIG. 14 (see example7). Based on these sequences, improved forward primers were designed,that may preferentially be used instead of primer M1F, in combinationwith reverse primer M1R These primers are: VAMSFb (forward) (SEQ ID NO23) VAMSFc (forward) (SEQ ID NO 24) VAMSFd (forward) (SEQ ID NO 25)VAMSFe (forward) (SEQ ID NO 26)

[0073] The sequence of these primers is shown in table 1. In order toobtain amplification of polynucleic acids from a maximal number ofisolates, primers VAMSFb, VAMSFc, VAMSFd and VAMSFe should be combinedin one PCR reaction.

[0074] According to a preferred embodiment, the present invention alsorelates to a method as defined above wherein step (iii) consists ofhybridizing the polynucleic acids obtained in step (ii) with a set ofprobes, under appropriate hybridization and wash conditions, said set ofprobes being preferentially applicable in a simultaneous hybridisationassay and comprising at least one probe hybridizing to a conservedregion of the cagA gene of H.pylori and at least one probe hybridizingto a variable region of the vacA gene of H.pylori, and morepreferentially said set of probes comprising at least one of thefollowing cagA- and vacA-derived probes as defined in Table 2 and inFIGS. 2 to 3: cag A-derived probe(s): cagApro (SEQ ID NO 1) cagprobe3(SEQ ID NO 27) vacA-derived probe(s): P1S1 (SEQ ID NO 2) P22S1a (SEQ IDNO 3) P1S1b (SEQ ID NO 4) P2S1b (SEQ ID NO 5) P1S2 (VAS2) (SEQ ID NO 6)P2S2 (SEQ ID NO 7) P1M1 (SEQ ID NO 8) P2M1 (SEQ ID NO 9) P1M2 (SEQ ID NO10) P2M2 (SEQ ID NO 11) P3S1 (SEQ ID NO 28) P4S1 (SEQ ID NO 29) P1M1new(SEQ ID NO 30) P2M1new (SEQ ID NO 31) P1M2new (SEQ ID NO 32) P2M2new(SEQ ID NO 33) P1M3 (SEQ ID NO 34)

[0075] or sequence variants thereof, with said sequence variantscontaining deletions and/or insertions and/or substitutions of one ormore nucleotides, mainly at their extremities (either 3′ or 5′), and orsubstitutions of non-essential nucleotides, —being nucleotides notessential in discriminating between alleles—, by others (includingmodified nucleotides such as inosine), or with said variants consistingof the complement of any of the above-mentioned oligonucleotide probes,or with said variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that the variants can hybridizespecifically with the same specificity as the oligonucleotide probesfrom which they are derived.

[0076] Probe cagApro was derived from published sequences of cagAalleles (Covacci et al., 1993; Tummuru et al. 1993). Based on theabove-mentioned novel sequences of the cagA gene (FIG. 10), provided bythe present invention, an improved probe was designed: cagprobe3. (SEQID NO 27)

[0077] The sequence of this probe is shown in table 2.

[0078] Probes P1S1, P22S1a, P1S1b, P2S1b, P1S2 and P2S2 are based on thesequences of the S-region of the vacA gene (FIG. 2), provided by thepresent invention These probes are designed to recognize sequences ofs1a, s1b and s2 variants, respectively. In a second instance, a largercollection of sequences of the S-region of the vacA gene is disclosed bythe present invention, as shown in FIG. 12 (see also example 6). Studyof the alignment of these novel sequences, as well as phylogeneticanalysis (FIG. 13), reveals the existence of a formerly unknown s1variant, in addition to the known variants s1a and s1b. This formerlyunknown variant is disclosed by the present invention and is denoteds1c. The present invention also provides novel probes, that permitspecific hybridization to the sic variant. These probe are: P3s1 (SEQ IDNO 28) P4s1. (SEQ ID NO 29)

[0079] The sequence of these probes is shown in table 2.

[0080] Probes P1M1, P2M1, P1M2 and P2M2 are based on the sequences ofthe M-region of the vacA gene that are provided by the present inventionand that are shown in FIG. 3. These probes are designed for specifichybridization to the m1 and m2 variants. Alignment of a larger number ofsequences of the M-region, also provided by the present invention,reveals the presence of 3 sequences that are different from the m1 andm2 variants (FIG. 14), as shown in example 7. These sequences mayrepresent a novel variant in the M-region. According to the presentinvention, this variant is denoted m3. Based on the sequences of theM-region that are shown in FIG. 14, novel probes have been designed,these probes being: P1M1new (SEQ ID NO 30) P2M1new (SEQ ID NO 31)P1M2new (SEQ ID NO 32) P2M2new (SEQ ID NO 33)

[0081] Probes P1M1 new and P2M1 new improve upon probes P1M1 and P2M1 inthat they are capable, when used together, to specifically hybridize toall ml sequences shown in FIG. 14. likewise, probes P1M2 new and P2M2new are improved probes that specifically hybridize to all m2 sequencesshown in FIG. 14. In addition, a novel probe that specificallyhybridizes to the aforementioned m3 sequences. is provided This probeis: P1M3. (SEQ ID NO 34)

[0082] The sequences of probes P1M1 new, P2M1 new, P1M2 new, P2M2 newand P1M3 are shown in table 2.

[0083] According to another embodiment, the present invention relates toa method for the detection of H.pylori strains present in a samplecomprising the steps of:

[0084] (i) if need be releasing, isolating or concentrating thepolynucleic acids in the sample;

[0085] (ii) amplifying the polynucleic acids of a relevant target regionof the vacA gene with a suitable primer pair, said primer pair beinggenerally applicable on different H.pylori strains, allowing to amplifysaid relevant target region of the vacA gene preferentially incompatible amplification conditions;

[0086] (iii) hybridizing the polynucleic acids obtained in (i) or (ii)with at least one probe hybridizing to a conserved region of the vacAgene;

[0087] (iv) detecting the hybrids formed in step (iii);

[0088] (v) determining the presence or absence of H.pylori in a samplefrom the hybridization signals obtained in step (iv).

[0089] Said method is referred to below as the “detection method”.

[0090] According to a preferred embodiment, the present inventionrelates to a method according to the preceding embodiment, wherein step(ii) consists of amplifying the polynucleic acids of a relevant targetregion in the vacA gene with suitable primers, said primers beinggenerally applicable on different H. pylori strains, allowing to amplifysaid relevant target region in compatible amplification conditions, withsaid target region being a conserved region, with said primerspreferentially being VA1F and VAL1X (SEQ ID NO14), or sequence variantsthereof, with said sequence variants containing deletions and/orinsertions and/or substitutions of one or more nucleotides, mainly attheir extremities (either 3′ or 5′), and or substitutions ofnon-essential nucleotides, —being nucleotides not essential indiscriminating between alleles, by others (including modifiednucleotides such as inosine), or with said variants consisting ofribonucleotides instead of deoxyribonucleotides, all provided that thevariants can hybridize/amplify specifically with the same specificity asthe oligonucleotide primers from which they are derived.

[0091] According to an even more preferred embodiment, the presentinvention relates to a method according to any of the two precedingembodiments, wherein step (iii) consists of hybridizing the polynucleicacids obtained in step (ii) with a set of probes, under appropriatehybridization and wash conditions, said set of probes beingpreferentially applicable in a simultaneous hybridisation assay andcomprising at least one probe hybridizing to a conserved region of thevacA gene of H.pylori, and more preferentially said set of probescomprising wing at least one of the following vacA-derived probes:HpdiaS1 (SEQ ID NO 35) HpdiaS2 (SEQ ID NO 36) HpdiaS3 (SEQ ID NO 37)HpdiaS4 (SEQ ID NO 38) HpdiaS5 (SEQ ID NO 39)

[0092] or sequence variants thereof with said sequence variantscontaining deletions and/or insertions and/or substitutions of one ormore nucleotides, mainly at their extremities (either 3′ or 5′), and orsubstitutions of non-essential nucleotides, —being nucleotides notessential in discriminating between alleles—, by others (includingmodified nucleotides such as inosine), or with said variants consistingof the complement of an, of the above-mentioned oligonucleotide probes,or with said variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that the variants can hybridizespecifically with the same specificity as the oligonucleotide probesfrom which they are derived.

[0093] According to another embodiment, the present invention relates toa probe composition for use in any detection/typing method as definedabove, said composition comprising at least one probe hybridizing to aconserved region of a VDG of H.pylori, and at least one probehybridizing to a variable region of vacA, and more preferentially saidprobes being derived from the polynucleic acid sequences of the vacAand/or cagA gene of H.pylori, and most preferentially said probes beenchosen from SEQ ID NO 1 to 11 and 27 to 34, or sequence variants thereofwith said sequence variants containing deletions and/or insertionsand/or substitutions of one or more nucleotides, mainly at theirextremities (either 3′ or 5′), and or substitutions of non-essentialnucleotides, —being nucleotides not essential in discriminating betweenalleles—, by others (including modified nucleotides such as inosine), orwith said variants consisting of the complement of any of theabove-mentioned oligonucleotide probes, or with said variants consistingof ribonucleotides instead of deoxyribonucleotides, all provided thatthe variants can hybridize specifically with the same specificity as theoligonucleotide probes from which they are derived.

[0094] According to another embodiment, the present invention relates toa probe composition for use in any detection method as defined above,said composition comprising at least one probe hybridizing to aconserved region of the vacA gene of H.pylori, and most preferentiallysaid probe being chosen from SEQ ID NO 35 to 39, or sequence variantsthereof with said sequence variants containing deletions and/orinsertions and/or substitutions of one or more nucleotides, mainly attheir extremities (either 3′ or 5′), and or substitutions ofnon-essential nucleotides, —being nucleotides not essential indiscriminating between alleles—, by others (including modifiednucleotides such as inosine), or with said variants consisting of thecomplement of any of the above-mentioned oligonucleotide probes, or withsaid variants coning of ribonucleotides instead of deoxyribonucleotides;all provided that the variants can hybridize specifically with the samespecificity as the oligonucleotide probes from which they are derived.

[0095] According to another embodiment, the present invention relates toa composition comprising at least one suitable oligonucleotideamplification primer, allowing to amplify the polynucleic acids of therelevant target regions of the respective VDG, said suitable primersbeing generally applicable with different H.pyiori strains and allowingthe amplification of said relevant target regions to be used incompatible amplification conditions, and more preferentially saidprimers allowing the amplification of a conserved region of the cagAgene and a region of the vacA gene comprising conserved and/or variabletarget regions, and most preferentially said primers being selected fromSEQ ID NO 12 to 26, or sequence variants thereof with said sequencevariants containing deletions and/or insertions and/or substitutions ofone or more nucleotides, mainly at their extremities (either 3′ or 5′),and or substitutions of non-essential nucleotides, —being nucleotidesnot essential in discriminating between alleles—, by others (includingmodified nucleotides such as inosine), or with said variants consistingof the complement of any of the above-mentioned oligonucleotide primers,or with said variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that the variants can hybridizespecifically with the same specificity as the oligonucleotide primersfrom which they are derived.

[0096] According to an even more specific embodiment the presentinvention relates to a probe being derived from the polynucleic acidsequences of the vacA and/or cagA gene of H.pylori, and with said probebeing chosen from SEQ ID NO 1 to 11 and 27 to 39, or sequence variantsthereon with said sequence variants containing deletions and/orinsertions and/or substitutions of one or more nucleotides, mainly attheir extremities (either 3′ or 5′), and or substitutions ofnon-essential nucleotides, —being nucleotides not essential indiscriminating between alleles—, by others (including modifiednucleotides such as inosine), or with said variants consisting of thecomplement of any of the above-mentioned oligonucleotide probes, or withsaid variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that the variants can hybridizespecifically with the same specificity as the oligonucleotide probesfrom which they are derived.

[0097] According to yet another even more preferred embodiment, thepresent invention relates to an oligonucleotide amplification primerallowing the amplification of a region of the cagA gene or a region ofthe vacA gene of H.pylori, and with said primer being selected from SEQID NO 12 to 26, or sequence variants thereof with said sequence variantscontaining deletions and/or insertions and/or substitutions of one ormore nucleotides, mainly at their extremities (either 3′ or 5′), and orsubstitutions of non-essential nucleotides, —being nucleotides notessential in discriminating between alleles—, by others (includingmodified nucleotides such as inosine), or with said variants consistingof the complement of any of the above-mentioned oligonucleotide primers,or with said variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that the variants canhybridize/amplify specifically with the same specificity as theoligonucleotide primers from which they are derived.

[0098] According to another embodiment the present invention relates toa method as defined above for the detection and/or typing of alleles ofVDG of H.pylori, more preferentially alleles of the cagA and vacA geneof H.pylori, present in a sample using a set of probes and/or primersspecially designed to detect and/or to amplify and/or to type the saidalleles, with said probes and primers being defined above.

[0099] According to another embodiment, the present invention relates toa method as defined above for the detection of alleles of VDG of H.pylori, more preferentially alleles of the vacA gene of H.pylori,present in a sample using a set of probes and/or primers speciallydesigned to detect and/or to amply the said alleles, with said probesand primers being defined above. In order to detect and/or type theH.pylori strains present in the sample, using the above set ofoligonucleotide probes, any hybridization method known in the art can beused (conventional dot-blot, Southern blot. sandwich, chip-based, etc).In order to obtain fast and easy results if a large number of probes isinvolved, a reverse hybridization format may be most convenient.According to a preferred embodiment, a selected set of probes areimmobilized onto a solid support.

[0100] In another preferred embodiment, a selected set of probes areimmobilized to a membrane strip. Said probes may be immobilizedindividually or as mixtures on the solid support. A specific and veryuser-friendly embodiment of the above-mentioned preferential method isthe LiPA-method, where the above-mentioned set of probes is immobilizedin parallel lines on a membrane. as further described in the examples.

[0101] Alternatively, detection—without typing—of H. pylori strains maybe performed conveniently by use of the DNA Enzyme Immuno Assay (DEIA).The principle of this assay as well as an application based on thedetection of a conserved part of the S-region of the vacA gene isoutlined in example 8.

[0102] Some of the above described probes are directed towards nucleicacid sequences already discribed in the prior art. However, asillustrated in the examples, nucleic acid sequences of VDG of a largenumber of new isolates of H.pylori were disclosed for the first time inthis invention, providing valuable new information necessary tosuccesfully design suitable probes with respect to detecting and moreimportantly to typing H.pylori strains. These new H. pylori sequencesalso form part of the present invention.

[0103] Moreover, previously designed primers and probes by other autors(Atherton et al, 1995) are shown in the examples to be less appropriatein typing H.pylori strains in a sample.

[0104] This invention also provides for probes and primers(sets) whichare designed to specifically detect or amplify the respective VDGalleles of the new isolates, and provides moreover methods and kits forapplying said primers or probes in the detection and/or typing ofH.pylori strains in a sample.

[0105] The present invention also provides for a set of primers,allowing amplification of the conserved region spanning the regionbetween the nucleotide at position 1 to the nucleotide at position 250of the cag gene of H.pylori. The set of primers comprises for instance:

[0106] cagf and cagr (SEQ ID N° 11 and 12)

[0107] Also, the present invention provides sets of primers covering thevariable S- and/or M-regions of the vacA gene of H.pylori, said S-regionbeing comprised between the nucleotide at position 1 and 300 andcomprising conserved sequences in addition to variable sequences, saidM-region being comprised between the nucleotides at the position 1450and 1650, with said primers for instance being:

[0108] VA1-F and VA1-XR (Atherton et al., 1995 and SEQ ID N° 15) M1F andM1R (SEQ ID N° 16 and 17)

[0109] The invention also provides methods and kits to apply the abovedescribed primers sets directed to particular regions of VDG genes, e.gthe cagA and vacA genes, simultaneously under identical amplification,hybridisation and washing conditions.

[0110] The primers according to the present invention may be labeledwith a label of choice (e.g. biotine). Different target amplificationsystems may be used, and preferentially PCR-amplification, as set out inthe examples. Single-round or nested PCR may be used.

[0111] According to yet another embodiment, the present inventionrelates to a solid support, preferentially a membrane strip, carrying onits surface, at least one probe as defined above. According to anotherembodiment, the present invention relates to a kit for detecting and/ortyping H. pylori strains in a sample liable to contain it, comprisingthe following components:

[0112] when appropriate at least one oligonucleotide primer as defined;

[0113] at least one probe as defined above, with said probe and/or otherprobes applied being by preference immobilized on a solid support;

[0114] a buffer or components necessary to produce the buffer enablingan amplification or a hybridization reaction between these probes andthe amplified products;

[0115] when appropriate a means for detecting the hybrids resulting fromthe preceding hybridization

[0116] The term “hybridization buffer” means a buffer enabling ahybridization reaction to occur between the probes and the polynucleicacids present in the sample, or the amplified products, under theappropriate stringency conditions.

[0117] The term “washing solution” means a solution enabling washing ofthe hybrids formed under the appropriate stringency conditions.

[0118] The present invention also relates to isolated vacA polynucleicacid sequences defined by SEQ ID NO 40 to 91 and SEQ ID NO 115 to 276 orany fragment thereof that can be used as a primer or as a probe in amethod for detection and/or typing of one or more vacA alleles of H.pylori.

[0119] The present invention also relates to isolated cagA polynucleicacid sequences defined by SEQ ID NO 92 to 114 or any fragment thereof,that can be used as a primer or as a probe in a method for detectionand/or typing of one or more cagA alleles of H. pylori.

[0120] The present invention also relates to a vacA protein fragmentencoded by any of the nucleic acids with SEQ ID NO 40 to 91 and SEQ IDNO 115 to 276 or any subfragment of said vacA protein fragment, withsaid subfragment consisting of at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acidsof a vacA protein.

[0121] The present invention also relates to a cagA protein fragmentencoded by any of the nucleic acids with SEQ ID NO 92 to 114, or anysubfragment of said cagA protein fragment, with said subfragmentcontisting of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a cagAprotein.

[0122] The following examples serve to illustrate the present inventionand are in no way to be construed as liming the scope of this invention.It should also be noted that the contents of all references referred toin this invention are hereby incorporated by reference.

LEGENDS TO TIRE FIGURES

[0123]FIG. 1: Schematic overview of the S- and M-region of the vacA geneof H.pylori and indication of the overall position of the relevantprimers.

[0124]FIG. 2a: DNA sequence alignment of the S-region S1a/b of variousH.pylori strains.

[0125]FIG. 2b: DNA sequence alignment of the S-region S2 of variousH.pylori strains.

[0126]FIG. 3a: DNA sequence alignment of the M-region M1 of variousH.pylori strains.

[0127]FIG. 3b: DNA sequence alignment of the M-region M2 of variousH.pylori strains.

[0128]FIG. 4: Agarose gel-electrophoresis of the amplification productsusing as starting material DNA from the gastric biopsy 18 and primersindicated in example 1.

[0129]FIG. 5: Agarose gel-electrophoresis of the amplification productsusing as starting material DNA from the gastric biopsy 41 and primersindicated in example 1.

[0130]FIG. 6: Agarose gel-electrophoresis of the amplification productsusing as starting material DNA from the gastric biopsy F67 and primersindicated in example 1.

[0131]FIG. 7: Agarose gel-electrophoresis of the amplification productsusing as starting material DNA from the gastric biopsy 25 and primersindicated in example 1.

[0132]FIG. 8: LIPA outline where the probes indicated in the figure areaccording to table II and primers according to example 3.

[0133]FIG. 9: Multiplex PCR with vacA as well as cagA primers. For vacAprimer set G was used (FIG. 1); for cagA primers cagf and cagr wereused. The isolate shown in the first two lanes contains s1 and m1alleles and is caga+. The isolate shown in lanes 4 and 5 (counting fromleft) contains a multiple infection.

[0134]FIG. 10: Alignment of cagA nucleic acid sequences, encoding theN-terminus of the cagA protein. The position of some cagA primers isindicated. Hyphens indicate gaps introduced to obtain optimal alignment.Asterisks below the alignment indicate identical nucleotides. Dots belowthe alignment indicate partial conservation.

[0135]FIG. 11: Phylogenetic tree of cagA amino acid sequences. The 16sequences counting from the top represent the first variant, occurringmainly in Europe and in Australia. USA123 and USA39 are stains from theUSA, having an intermediate position. The 7 sequences counting from thebottom (HK7 to HKTh8828) represent a variant that is mainly found in FarEast Asia.

[0136]FIG. 12: Alignment of nucleic acid sequences of part of theS-region of the vacA gene. The sequences are grouped according to thevariant that they belong to. A larger number of sequences is shown thein FIGS. 2a and 2 b. The variants are from top to bottom; s2, s1c, s1band s1a. Hyphens indicate that at that position the nucleotide isidentical to that in the sequence of stain 29401. Dots indicate a gap inthe sequence that was introduced to preserve alignment.

[0137]FIG. 13: Phylogenetic analysis of nucleic acid sequences of partof the S-region of the vacA protein. The variants are indicated.

[0138]FIG. 14: Alignment of nucleic acid sequences of part of theM-region of the vacA gene. A larger number of sequences is shown than inFIGS. 3a and 3 b. Hyphens and dots as in FIG. 12.

[0139]FIG. 15: Phylogenetic a of nucleic acid sequences of part of theM-region of the vacA protein The variants are indicated.

EXAMPLES Example 1 Evaluation of the Use of the Primers Described byAtherton et al., 1995 in typing H.pylori Strains within the Framework ofLarge Scale Clinical Trials

[0140] 1.1 Comparison of VacA Genotyping Methods.

[0141] The efficiency of the vacA genotyping as described by Atherton etal. (1995) was compared to the efficacy as described in the presentinvention. The method as described by Atherton comprises 6 different PCRreactions:

[0142] A. Using primers VA1F and VA1R, to distinguish s1 and s2 alleles.

[0143] B. Using primers SS1F and VA1R, to amplify s1a sequences.

[0144] C. Using primers SS3F and VA1R, to amplify s1b sequences.

[0145] D. Using primers SS2F and VA1R, to amplify s2 sequences.

[0146] E. Using primers VA3F and VA3R, to amplify m1 sequences.

[0147] F. Using primers VA4F and VA4R, to amplify m2 sequences.

[0148]FIG. 1 shows a schematic representation of all primers involved invacA 1 ping. Identification of the PCR products is based on visualinspection of DNA bands on an agarose gel.

[0149] 1.2. Problems with the Atherton System:

[0150] The s-region:

[0151] Based on the sequence alignments from European isolates. as shownin FIGS. 2a and 2 b it is clear that primers SS1F, SS2F, and SS3F maycontain several mismatches to their respective target sequences. Thismay hamper proper annealing of the primers and may lead to amplificationof spurious bands. The target sequence for primer SS3F (aimed atdetection of the s1b allele). contains two crucial mismatches at the 3′end of the primer in some isolates (e.g. in isolates F67, F68, F73, F76,F42, F12).

[0152] F67 (see below) showed amplification with primer SS1F and VA1R,whereas amplification with SS3F and VA1R was negative, suggesting thepresence of the s1a genotype. However, PCR/LiPA analysis showed thepresence of genotype s1a, which was confirmed by sequence analysis.

[0153] Primer SS2F, aimed at s2 sequences, results in amplification of aspecific bands (see e.g. figure photo 1 & 2, in case of primerset D).

[0154] The m-region:

[0155] As described by Atherton el al., (1995) typing of the m-regionwas initially based on hybridization with two specific DNA probes, ie.pCTB4 and VA6 for the M1 and M2 variant, respectively.

[0156] From the published nucleotide alignments of the vacA sequencesfrom strain 60190 (type M1) and Tx30a (U29401; type M2). it is obviousthat these two probes cover a region of substantial variation.

[0157] Moreover, the M1 variant shows a deletion (around position 2340of the 60190 sequence), compared to the M2 variant. One might envisagethat this region of deletion/insertion (or to the S-region) is of majorimportance to discriminate M1 and M2. However, the PCR primers forspecific detection of M1 and M2 are aimed at a different region of thevacA gene, which is more downstream (between positions 2750 and 3030 ofthe 60190 sequence)and is not covered by the original DNA probes.

[0158] We have analysed the individual PCR primers by sequencealignments to the M1 and M2 sequences. We noticed that the 3′ ends ofseveral primers described by Atherton et al., are not completely uniquein the vacA gene.

[0159] Primer VA3-R shows homology to sequences:

[0160] in strain 60190 (Genbank Seq U05676; m1-type):

[0161] around pos 229 (6 nt at the 3′ end)

[0162] around pos 839 (6 nt at the 3′ end)

[0163] around pos 3011 (target sequence, 100%)

[0164] around pos 4653 (6 nt at the 3′ end)

[0165] in strain Tx30a:

[0166] around pos 4271 (6 nt at the 3′end)

[0167] Primer VA4-F shows homology to sequences:

[0168] in strain Tx30a (GenBank Seq # U29401; m2-type):

[0169] around pos 231 (7 nt at the 3′ end)

[0170] around pos 1907 (8 nt at the 3′ end)

[0171] around pos 2297 (target sequence, 100%)

[0172] around pos 2594 (9 nt at the 3′ end)

[0173] Especially the homologies at the very 3′ end may hamper thespecificity of these primers. Some spurious bands were obtained whenusing these primers. Moreover, these primers failed to yield anyamplification product in several isolates or biopsies (e.g., biopsy 41,see below). This has been observed before (Maeda, S, K Ogura. M. IshitomF. Kanai H. Yoshida, S. Ota, Y. Shiratori, and M. Omata. abstract # 492:Diversity of Helicobacter pylori E acA gene in Japanese strains —highcytotoxin activity type s1 is dominant in Japan, Digestive Disease WeeksSan Fransisco, May 1996).

[0174] We have analysed the M1 and M2 region of be vacA allele frommultiple H.pylori strains by DNA sequencing upon PCR amplification usingas primers HPMGF and HPMGR (see FIGS. 3a and 3 b). Based on thesesequences new primers had to be developed for vacA genotyping in amultiplex PCR, as described in example 4.

[0175] 1.3. Comparative Results are Shown in Figures and DiscussedBelow:

[0176] The respective primers used by Atherton et al. (1995) were usedin A-F while primerset G. comprised the newly designed set of primerscomprising VA1F, VA1XR, M1F, and M1R disclosed for the first time inthis invention. AL of these primers are new as such, except VA1F whichwas disclosed by Atherton et al., 1995.

[0177] Biopsy #18 (see FIG. 4) A s1/s2 S1 B s1a + C s1b − D s2 − (notethe background) E m1 − F m2 + G multi s1/m2

[0178] From this biopsy, the expected fragments were amplified,consistent with a s1a/m2 genotype. Multiplex PCR followed by LiPA asdescribed in the present invention, yielded an identical result.

[0179] Biopsy #41 (see FIG. 5) A s1/s2 s1 B s1a + C s1b − D s2 − (notethe background) E m1 − F m2 − G multi s1/m1

[0180] From this biopsy, only the s-region could be typed by the methodof Atherton et al. Amplification with the m1 and m2-specific primers didnot yield any visible DNA product. However, by the multiplex PCRfollowed by LIPA as described in the present invention. a s1a, m1genotype was detected.

[0181] Isolate F67 (see FIG. 6) A s1/s2 s1 B s1a − C s1b − D s2 ÷ (notethe background) E m1 − F m2 − G multi s1/m1

[0182] LiPA showed the presence of a s1b, instead of s1a. This wasconfirmed by sequence analysis,

[0183] 1.4, Detection of Multiple Infections

[0184] Biopsy 25 (see FIG. 7) A s1/s2 both B s1a − C s1b + D s2 + (notethe background) E m1 + F m2 + G multi s1/s2/m1/m2

[0185] LiPA analysis revealed the presence of s1b/s2/m1/m2 mixedgenotypes.

Example 2 Identification and Amplification of a Conserved Region of thecagA Gene Fragment in H.pylori; Designing Primers and a cagA-DerivedProbe Allowing to Detect H.pylori in a Sample Through ReverseHybridization

[0186] The establishment of the experimental conditions in order to setup a reverse hybridisation assay in case of the cagA gene comprised i) atheoretical evaluation of suitable probes and primers based upon nucleicacid sequence comparisons using standard DNA analysing computerprogrammes, and ii) an experimental evaluation and adjustment of theprimers and probes to the conditions set for the reverse hybridizationtechnology.

[0187] Comparison of two published nucleic acid sequences of cagAalleles of different H.pylori strains demonstrated that the regionbetween nucleic acids 17 to 113 is highly conserved (Covacci et al.,1993; Tummuru et al, 1993) and said region could be used for positiveidentification of the presence of the cagA gene in a certain H.pyloristrain.

[0188] A set of primers was designed as follows: cagF (bp 17 to 40) cagR(bp 178-199)

[0189] Both primers are new primer sequences, described by the currentinvention (see table I). These primers can be labeled with a label ofchoice (e.g. biotine). Different primer-based target-amplificationsystems may be used. For amplification using the PCR, the conditionsused in case of a single-round amplification with above primers cagF andcagR involve 40 cycles of 1 min/95° C., 1 min/55° C. 1 min/72° C.followed by a final extension for 5 min at 72° C.

[0190] The PCR reaction mixture was as follows:

[0191] 1 μI DNA sample containing H.pylori or control DNA

[0192] 10 μl 10×polymerase mix (final concentration 10 m Tris HCl pH19.0, 50 mM KCl 2.5 mM MgCl, 0.01% gelatin, and 0.1% Triton)

[0193] 20 μl deoxyribonucleotide mix (final concentration 200 μM each)

[0194] 1 μl Super Taq polymerase (0.25 U/μl)

[0195] 1 μl forward primer (50 pmole(μl)

[0196] 1 μl reverse primer (50 pmoles/μl)

[0197] 66 μl water

[0198] 100 μl

[0199] Amplification products were analysed on an agarose gel, stainedwith ethidiumbromide and visualized under UV. The amplified productobtained using 1 μl of H.pylori DNA as starting material and aboveprimers consisted of a single band with approximatively molecular weight0.18 Kb, in agreement with the expected size of 183 bp. Control samplescontaining DNA from cag(−) H.pylori strains or other bacterial speciesdid not yield any amplification product (data not shown).

[0200] The uniformity of the amplified product was verified through DNAsequencing applying standard sequencing techniques. A 100% match withthe described region could be demonstrated (data not shown).

[0201] Also, a number of probes were tested in order to determineoptimal hybridization between the above amplified product and the saidprobes under standardized hybridization and washing conditions appliedin the reverse hybridisation assay.

[0202] The below probes tested were chosen from the list indicated intable II. Said probes were immobilized onto a solid support as describedin example 3. The amplified product obtained with said above primers washybridized to the respective probes applying the same conditions asoutlined in example 3. Most optimal results were obtained with probecagApro (SEQ ID N° 12), which can thus be used as a positiveidentification of the presence of the cagA gene in H. pylori strains, incombination with the above primers under the conditions of the belowreverse hybridization assay.

Example 3 Identification and Amplification of Variable Target Regions ofthe vacA Gene in H.pylori; Designing Primers and a vacA-Derived ProbeAllowing to Detect and/or Type H.pylori in a Sample Through ReverseHybridization

[0203] The establishment of the experimental conditions in order to setup a reverse hybridisation assay in case of the vacA gene comprised i) atheoretical evaluation of suitable probes and primers based upon nucleicacid sequence comparisons using standard DNA analysing computerprogrammes, and ii) an experimental amplification of the variousvariable regions, DNA sequence analysis of the respective amplifiedfragments, designing allele-specific probes and appropriate primers, andthe evaluation and the adjustment of the primers and probes to theconditions applicable in the reverse hybridization technology.

[0204] Recently Atherton et al. (1995) demonstrated the presence of twovariable regions in the vacA gene, being the S- and M-region. Primerswere designed in order to amplificate specifically alleles of the vacAwith variable S- and M-regions.

[0205] In this invention, a large number of additional nucleic acidsequences spanning both the said S- or M-region were obtained upon DNAsequence analysis of PCR amplification of said regions. These data arenew and are being disclosed here for the first time in the presentinvention (see FIGS. 2 and 3).

[0206] In order to obtain amplification products spanning either the S-or the M-region of the vacA gene, the following set of primers was used:S-region: VA1-F (see Atherton et al., 1995) VA1-R (see Atherton et al.,1995) M-region: HPMGF (CACAGCCACTTTCAATAACGA) HPMGR(CGTCAAAATAATTCCAAGGG)

[0207] These primers can be labeled with a label of choice (in this casebiotine was used). Different primer-based target-amplification systemsmay be used. For amplification using the PCR the conditions used in caseof a single-round amplification with above primers, involve 40 cycles of1 min/95° C., 1 min/55° C. 1 min/72° C. followed by a final extensionfor 5 min at 72° C.

[0208] The PCR reaction mixture was as follows:

[0209] 1 μl DNA sample, containing H.pylori or control DNA

[0210] 10 μl 10×polymerase mix (final concentration 10 mM Tris HCl, pH9.0, 50 mM KCl 2.5 mM MgCl₂, 0.01% gelatin, and 0.1% Triton)

[0211] 20 μl deoxyribonucleotide mix (final concentration 200 μM each)

[0212] 1 μl Super Taq polymerase (0.25 U/μl)

[0213] 1 μl forward primer (50 pmoles/μl)

[0214] 1 μl reverse primer (50 pmoles/μl)

[0215] 66 μl water

[0216] 100 μl

[0217] Amplification products were analysed by DNA sequencing applyingstandard sequencing techniques. The results of these analyses are givenin FIGS. 2 and 3. Based on these analyses, it became obvious thatprimers being used by others with the aim of allele-specific typing ofH.pylori based upon the variable S- and M-region of the vacA gene, couldnot cover the full range of pathogenic H.pylori strains (see example 1).Thus, new sets of primers, not obvious to the skilled man in the art,were designed in order to develop an assay to detect and type pathogenicH.pylori strains in a sample. The primers and their sequence are givenin table I. Also, a number of probes were tested in order to obtainoptimal hybridization between the amplified products, generated by thenew primer sets, and said probes under standardized hybridization andwashing conditions applied in the reverse hybridisation assay. Thetested probes are given in table II. Said probes were immobilized unto asolid support as described by Styver et al., 1993. The amplificationwith said primersets was performed under the conditions and protocol asdescribed above in this example. The amplified products obtained withsaid above primers were hybridized to the respective probes (see FIG.8).

[0218] Optimal results were obtained combining the following primers:VA1-F (Atherton et al., 1995) VA1XR (SEQ ID N014) M1F (SEQ ID NO15) M1R(SEQ ID N016)

[0219] with the following probes: P1S1 (SEQ ID NO2) P22S1a (SEQ ID NO3)P1S1b (SEQ ID NO4) P2S1b (SEQ ID NO5) P1S2(VAS2) (SEQ ID NO6) P2S2 (SEQID NO7) P1M1 (SEQ ID NO8) P2M1 (SEQ ID NO9) P1M2 (SEQ ID NO10) P2M2 (SEQID NO11)

Example 4 Development of the Line Probe Assay (LiPA)-Strip

[0220] The principle and protocol of the line probe assay was in essenceas described earlier (Stuyver et al., 1993). Good results were obtainedcombining the following primers: cagF (SEQ ID NO 12) cagR (SEQ ID N0 13)VA1-F (Atheron et al., 1995) VA1XR (SEQ ID NO 14) M1F (SEQ ID NO 15) M1R(SEQ ID NO 16)

[0221] with the following probes: cagApro (SEQ ID NO 1) P1S1 (SEQ ID NO2) P22S1a (SEQ ID NO 3) P1S1b (SEQ ID NO 4) P2S2b (SEQ ID NO 5)P1S2(VAS2) (SEQ ID NO 6) P2S2 (SEQ ID NO 7) P1M1 (SEQ ID NO 8) P2M1 (SEQID NO 9) P1M2 (SEQ ID NO 10) P2M2 (SEQ ID NO 11)

[0222] The said primers were labeled with biotine. Differentprimer-based target-amplfication systems may be used. For amplificationusing the PCR, the conditions used in case of a single-roundamplification with above primers, involve 40 cycles of 1 min/95° C., 1min/55° C., 1 min/72° C. followed by a final extension for 5 min at 72°C.

[0223] The PCR reaction mixture was as follows:

[0224] 1 μl DNA sample, containing H.pylori or control DNA

[0225] 10 μl 10×polymerase mix (final concentration 10 mM Tris HCl, pH9.0, 50 mM KCl 2.5 mM MgCl₂, 0.01% gelatin and 0.1% Triton)

[0226] 20 μl deoxyribonucleotide mix (final concentration 200 μM each)

[0227] 1 μl Super Taq polymerase (0.25 U/μl)

[0228] 1 μl forward primer (50 pmoles/μl)

[0229] 1 μl reverse primer (50 pmoles/μl)

[0230] 66 μl water

[0231] 100 μl

[0232] The sequence of these primers is given in table 2. An example ofthe amplification products generated by use of vacA s/m region-primersor cagA-primers is shown in FIG. 9. For this experiment primer set G(FIG. 1) was used for vacA and cagf and cagr were used for cagA. Theisolate shown in the first two lanes contains s1 and m1 alleles and iscagA+. The isolate shown in lanes 4 and 5 (counting from left) containsa multiple infection. The results of the LiPA are shown in FIG. 8.

Example 5 Novel DNA Sequences of a Fragment of the cagA Gene of H.pylori and Design of Primers and a Probe Based Thereon.

[0233] The 5′ part of the cagA gene was amplified by PCR from various H.pylori isolates, using different primer combinations. The resultingfragments were sequenced and the alignment is shown in FIG. 10. Thesequences comprised 449-464 bp, starting at the start codon of the ORF.A total of 149-154 amino acids representing the N-terminus of the cagAprotein can be derived by translation of these sequences, sting at theATG codon at position 1 in FIG. 10.

[0234] As shown by phylogenetic analysis in FIG. 11, 2 different formsof cagA were recognized. The first variant is highly homologous to thereference sequence (Genbank accession number L11741 (HECMAJANT) orX70039 (HPCAI)) and occurs mainly in strains from Europe and Australia.Two sequences from the USA (J123 and J39) seem to have intermediatepositions in the phylogenetic tree. The second variant, mainly found instrains from Far East Asia. contains 15 additional nucleotides betweennt positions 20 and 31, encoding 5 additional amino acids betweenpositions 8-9, as compared to the reference sequence.

[0235] From the nucleotide sequence alignment the following novelprimers and probe were deduced aimed at highly conserved regions in thecagA gene. TABLE 3 CagA primers and probe position/ primer/probe 5′to3′sequence orientation¹ primers cagFN1 GATAAGAAYGATAGGGATAA +(142-161)cagRN1 AATACTGATTCTTTTTGG −(230-247) probe cagprobe3GGATTTTTGATCGCTTTATT −(219-227)

Example 6 Novel DNA Sequences of the s-Region of the vacA Gene of H.pylori and Design of Probes Based Thereon.

[0236] VacA s-region fragments were amplified from a large number of H.pylori isolates, using primers VA1-F and VA1-R (Atherton et al., 1995).This resulted in fragments of 176 bp for s1 and 203 bp for s2 typessequences. Parts of these fragments were sequenced, and the resultingalignment of 80 sequences (including 2 reference sequences U29401 andU07145) is shown m FIG. 12. Apart from the already known s1a and s1btype sequences, a third variant was observed, mainly in isolates fromFar East Asia (Japan, China. Hong Kong), This variant is designated s1c.Type s1c has several highly consistent mutations as compared to type s1band s1a. These mutations allow specific recognition of each of the s1subtypes. Phylogenetic analysis, as shown in FIG. 13, reveals distinctclusters of s1a, s1b, s1c and s2 sequences. The N-terminal parts of thevacA protein can be deduced from the nucleic acid sequences of the s1a,s1b, s1c, and s2 variants by translation starting at codon CCT atposition 2 in FIG. 12. This reveals the presence of a single conservedamino acid imitation (Lys) at position 22 in subtype s1c as compared tos1a and/or s1b sequences. All other nucleotide mutations appear to besilent.

[0237] New probes were designed to specifically detect the s1c variants:P3s1: 5′ GGGYTATTGGTYAGCATCAC 3′ (positions 26-45) P4s1:5′ GCTTTAGTRGGGYTATTGGT 3′ (positions 17-36)

[0238] Thus, for optimal detection of the vacA s-region variants, thefollowing probes were used: for s1a: P1S1 and P22S1a for s1b: P1s1b andP2s1b for s2: P1S2 (VAS2) and P2S2 for s1c: P3s1 and P4s1

Example 7 Novel DNA Sequences of the m-region of the vacA Gene of H.pylori and Design of Probes Based Thereon.

[0239] The vacA m-region was analyzed from a number of H. pyloriisolates, by using primers BPMGF and HPMGR These primers allow generalamplification of larger parts of the m-region sequences and generatefragments of 401 and 476 bp for m1 and m2 variants, respectively.Fragments were sequenced and the alignment of 86 m-region sequences(including reference sequences U05677, U07145, U05676 and U29401) isshown in FIG. 14. The phylogenetic tree is shown in FIG. 15. Thealignment revealed the presence of 3 sequences (Ch4, Hk41, Hk46) thatare different from the published m1 and m2 variants. These sequences nayrepresent another variant in the m-region. Said new variant may bedenoted m3.

[0240] These alignments revealed that the target sequence for forwardprimer M1F (SEQ ID NO15) was not completely conserved among allisolates. The target sequence for reverse primer M1R appeared highlyconserved among all isolates. As an alternative for forward primer M1Fthe following primers were designed, as shown in table 4. TABLE 4 Novelforward primers for the vacA m-region primer sequence 5′to 3′orientation VAMSFb: GTGGATGCCCATACGOCTAA forward VAMSFcGTGGATGCTCATACAGCTWA forward VAMSFd GTGGATGCCCATACGATCAA forward VAMSFeGCGAGCGCTCATACGGTCAA forward

[0241] PCR amplification in the m-region of the vacA gene can thus beperformed by use of VAMSFb,c,d, and e as forward primers and M1R as thereverse primer.

[0242] Novel probes were designed for specific hybridization to m1 andm2 variants. Their sequence is based on the above-mentioned probes P1m1,P2m1, P1m2 and P2 m2. In order to obtain reactivity with all sequences,a few degeneracies were included. The novel sequences are shown in table5. For specific identification of mn3 variants, a single probe is added(P1m3). TABLE 5 Novel probes for the vacA m-region probe sequence 5′-3′positions P1m1new TTGATACKGGTAATGGTGG as for P1m1 P2m1newKGGTAATGGTGGTTTCAACA as for P2m1 P1m2new KGGTAATGGTGGTTTCAACA as forP1m2 P2m2new AGAGCGATAAYGGKCTAAACA as for P2m2 P1m3AGGGTAGAAATGOTATCGACA 1577-1597¹

Example 8 Detection of H. pylori DNA by PCR and DNA Enzyme Immuno Assay(DEIA).

[0243] This method is used for rapid and specific detection of PCRproducts. PCR products are generated by a primerset, of which either theforward or the reverse primer contain biotin at the 5′ end. This allowsbinding of the biotinylated amplimers to streptavidin-coated microtiterwells. PCR products are denatured by sodium hydroxide, which allowsremoval of the non-biotinylated strand Specificdigoxigenin(DIG)-labelled oligonucleotide probes are hybridized to thesingle-stranded immobilized PCR product and hybrids are detected byenzyme-labelled conjugate and colorimetric methods.

[0244] For detection of H. pylori DNA, the vacA s-region is used as atarget. PCR primers VA1F and biotinylated VA1XR are used for PCR of thevacA s-region. A multiplex PCR can be performed on the vacA s andm-regions. The result of PCR is then tested by the DEIA, using probesaimed at the s-region. In case of a positive result the same PCRmixture, including amplimers from both the vacA s- and m-regions, cansubsequently be used on a vacA LiPA

[0245] The PCR mixtures can be composed as follows: 1 μl target DNA 5 μl10 × PCR buffer (final concentration 10 mM TrisHCl pH 8.3, 50 mM KCl,1-3 mM MgCl₂) 10 μl 5 × dNTP's (1 nM) 0.3 μl AmpliTaq Gold DNApolymerase (5 units/μl) 1 μl VA1F (25 pmoles/μl) 1 μl VA1Xr (25pmoles/μl) 1 μl VAMSFb (25 pmoles/μl) 1 μl VAMSFc (25 pmoles/μl) 1 μlVAMSFd (25 pmoles/μl) 1 μl VAMSfe (25 pmoles/μl) 1 μl M1R (25 pmoles/μl)26.3 μl water 50 μl total

[0246] The following PCR program can be used:

[0247] 9 min pre-incubation at 94° C.

[0248] 40 cycles of 1 min 94° C., 1 min 50° C., and 1 min 72° C.

[0249] final extension: 5 min at 72° C.

[0250] The mixture of probes used for detection of the vacA s-region isshown in table 6. TABLE 6 Probes for detection of vacA s-regionamplimers by DEIA probe sequence target HpdiaS1DIG-CATGCYGCCTTCTTTACAACCGT s1 HpdiaS2 DIG-CATGCCGCCTTTTTCACRACCGT s1HpdiaS3 DIG-CATGCCGCTCTTTTTACAACCGT s1 HpdiaS4DIG-CATGCCGCCTTTTTTACAACCGT s1 HpdiaS5 DIG-AGTCGCGCYTTTTTYACAACCGT s2

[0251] Practically, microtiterplate we %s were precoated withstreptavidin. Ten μl of PCR product was mixed with amplimer dilutionbuffer (1×SSC, 0.1% Tween-20, and 0.004% phenol red). After incubationat 42° C. for 30 minutes, the wells were washed 3 times with 400 μlwashing solution (1×SSC, 0.1% Tween-20), The captured PCR products weredenatured by addition of 100 μl of 0.1M NaOH into the well and incubatedfor 5 minutes at room temp. The fluid, containing the unbiotinylatedeluted strand was removed. 100 μl hybridization solution containing1×SSC, 0.1% Tween-20, 0.004% phenol red and 1 pmole of digoxigenin (DIG)labelled oligonucleotide probe(s) were added to the well and incubatedfor 45 minutes in a waterbath at 42° C. After washing the wells 3 timeswith washing solution, 100 μl of 75mU/ml anti-digoxigenin-peroxidaseconjugate (Boehringer Mannheim) was added and incubated for 15 minutesin a waterbath at 42° C. The unbound conjugate was removed by washingthe wells 5 times with washing solution 100 μl of substrate solutioncontaining tetramethylbenzidine (TMB) was added to the wells. Afterincubation for 15 minutes at room temperature the colour reaction wasstopped by addition of 100 μl 0.5M sulphuric acid. The optical densityof the wells was read at 450 nm in a microtiter plate reader.

[0252] For interpretation of the results, optical densities of thesamples were compared with negative controls and borderline positivecontrols. Table 7 shows the result of a DEIA analysis of 6 samplesSample 1 and 5 yield an optical density that is higher than that of theborderline positive control, these samples are therefore consideredpositive. The optical density of the other samples is lower than theborderline positive control; they are considered negative. TABLE 7Results of a DEIA test Sample OD conclusion negative positive control1.178 borderline pos. control 0.214 negative control 0.102 sample 1 >4.0positive sample 2 0.086 negative sample 3 0.098 negative sample 4 0.108negative sample 5 2.146 positive sample 6 0.096 negative

[0253] TABLE 1 Nucleotide sequence of the primers: cag2F5′-TTGACCAACAACCACAAACCGAAG-3′ SEQ ID NO 12 cagR5′-CTTCCCTTAATTGCGAGATTCC-3′ SEQ ID NO 13 VA1-F5′-ATGGAAATACAACAAACACAC-3′ Atherton et al. 1995 VA1XR5′-CCTGARACCGTTCCTACAGC-3′ SEQ ID NO 14 M1F 5′-GTGGATGCYCATACRGCTWA-3′SEQ ID NO 15 M1R 5′-RTGAGCTTGTTGATATTGAC-3′ SEQ ID No 16 HPMGF5′-CACAGCCACTTTCAATAACGA-3′ SEQ ID No 17 HPMGR5′-CGTCAAAATAATTCCAAGGG-3′ SEQ ID No 18 cagSF 5′-CAACAACCACAAACCGAAG-3′SEQ ID No 19 cagSR 5′-GATTGGTTTTTGATCAGGATC-3′ SEQ ID No 20 cagFN15′-GATAAGAAYGATAGGGATAA-3′ SEQ ID No 21 cagRN1 5′-AATACTGATTCTTTTTGG-3SEQ ID No 22 VAMSFb GTGGATGCCCATACGGCTAA SEQ ID No 23 VAMSFcGTGGATGCTCATACAGCTWA SEQ ID No 24 VAMSFd GTGGATGCCCATACGATCAA SEQ ID No25 VAMSFe GCGAGCGCTCATACGGTCAA SEQ ID No 26

[0254] TABLE 2 Nudcotide sequence of the probes: cagApro SEQ ID NO 1GTTGATAACGCTGTCGCTTC (pos. 94-113) P1S1 SEQ ID NO 2 GGAGCRTTRGTCAGCATCAC(pos. 61-80 of vacA ORF of strain 60190 (Genbank Acc. U05676)) P22S1aSEQ ID NO 3 GCTTTAGTAGGAGCRTTRGTC (pos. 52-72 of vacA ORF of strain60190 (Genbank Acc. U05676)) P1S1b SEQ ID NO 4 GGAGCGTTGATTAGYKCCAT (pos61-80) P2S1b SEQ ID NO 5 GTTTTAGCAGGAGCGTTGA (pos 52-72) P1S2(VAS2) SEQID NO 6 GCTAAYACGCCAAAYGATCC (pos. 88-107 of vacA ORF of strain Tx30a(Genbank Acc. U29401)) P2S2 SEQ ID NO 7 GATCCCATACACAGCGAGAG (pos.103-122 of vacA ORF of strain Tx30a (Genbank Acc. U29401)) P1M1 SEQ IDNO 8 TTGATACGGGTAATGGTGG (pos. 1526-1544 of vacA ORF of strain 60190(Genbank Acc. U05676)) P2M1 SEQ ID NO 9 GGGTAATGGTGGTTTCAACA (pos.1533-1552 of vacA ORF of strain 60190 (Genbank Acc. U05676)) P1M2 SEQ IDNO 10 ACGAATTTAAGAGTGAATGGC (pos. 1522-1542 of vacA ORF of strain Tx30a(Genbank Acc. U29401)) P2M2 SEQ ID NO 11 AGAGCGATAACGGGCTAAACA (pos.1577-1597 of vacA ORF of strain Tx30a (Genbank Acc. U29401)) cagprobe3SEQ ID NO 27 GGATTTTTGATCGCTTTATT (pos. 219-227) P3S1 SEQ ID NO 28GGGYTATTGGTYAGCATCAC (pos. 26-45) P4S1 SEQ ID NO 29 GCTTTAGTRGGGYTATTGGT(pos. 17-36) P1M1new SEQ ID NO 30 TTGATACKGGTAATGGTGG P2M1new SEQ ID NO31 KGGTAATGGTGGTTTCAACA P1M2new SEQ ID NO 32 KGGTAATGGTGGTTTCAACAP2M2new SEQ ID NO 33 AGAGCGATAAYGGKCTAAACA P1M3 SEQ ID NO 34AGGGTAGAAATGGTATCGACA HpdiaS1 SEQ ID NO 35 DIG-CATGCYGCCTTCTTTACAACCGTHpdiaS2 SEQ ID NO 36 DIG-CATGCCGCCTTTTTCACRACCGT HpdiaS3 SEQ ID NO 37DIG-CATGCCGCTCTTTTTACAACCGT HpdiaS4 SEQ ID NO 38DIG-CATGCCGCCTTTTTTACAACCGT HpdiaS5 SEQ ID NO 39DTG-AGTCGCGCYTTTTTYACAACCGT

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1 280 1 20 DNA Artificial Sequence cagApro probe 1 gttgataacg ctgtcgcttc20 2 20 DNA Artificial Sequence P1S1 vacA-derived probe 2 ggagcrttrgtcagcatcac 20 3 21 DNA Artificial Sequence P22S1a vacA-derived probe 3gctttagtag gagcrttrgt c 21 4 20 DNA Artificial Sequence P1S1bvacA-derived probe 4 ggagcgttga ttagykccat 20 5 19 DNA ArtificialSequence P2S1b vacA-derived probe 5 gttttagcag gagcgttga 19 6 20 DNAArtificial Sequence P1S2(VAS2) vacA-derived probe 6 gctaayacgccaaaygatcc 20 7 20 DNA Artificial Sequence P2S2 vacA-derived probe 7gatcccatac acagcgagag 20 8 19 DNA Artificial Sequence P1M1 vacA-derivedprobe 8 ttgatacggg taatggtgg 19 9 20 DNA Artificial Sequence P2M1vacA-derived probe 9 gggtaatggt ggtttcaaca 20 10 21 DNA ArtificialSequence P1M2 vacA-derived probe 10 acgaatttaa gagtgaatgg c 21 11 21 DNAArtificial Sequence P2M2 vacA-derived probe 11 agagcgataa cgggctaaac a21 12 24 DNA Artificial Sequence cagF primer 12 ttgaccaaca accacaaaccgaag 24 13 22 DNA Artificial Sequence cagR primer 13 cttcccttaattgcgagatt cc 22 14 20 DNA Artificial Sequence VA1XR primer 14cctgaraccg ttcctacagc 20 15 20 DNA Artificial Sequence M1F primer 15gtggatgcyc atacrgctwa 20 16 20 DNA Artificial Sequence M1R primer 16rtgagcttgt tgatattgac 20 17 21 DNA Artificial Sequence HPMGF primer 17cacagccact ttcaataacg a 21 18 20 DNA Artificial Sequence HPMGR primer 18cgtcaaaata attccaaggg 20 19 19 DNA Artificial Sequence cagSF primer 19caacaaccac aaaccgaag 19 20 21 DNA Artificial Sequence cagSR primer 20gattggtttt tgatcaggat c 21 21 20 DNA Artificial Sequence cagFN1 primer21 gataagaayg atagggataa 20 22 18 DNA Artificial Sequence cagRN1 primer22 aatactgatt ctttttgg 18 23 20 DNA Artificial Sequence VAMSFb primer 23gtggatgccc atacggctaa 20 24 20 DNA Artificial Sequence VAMSFc primer 24gtggatgctc atacagctwa 20 25 20 DNA Artificial Sequence VAMSFd primer 25gtggatgccc atacgatcaa 20 26 20 DNA Artificial Sequence VAMSFe primer 26gcgagcgctc atacggtcaa 20 27 20 DNA Artificial Sequence cagprobe3cagA-derived probe 27 ggatttttga tcgctttatt 20 28 20 DNA ArtificialSequence P3S1 vacA-derived probe 28 gggytattgg tyagcatcac 20 29 20 DNAArtificial Sequence P4S1 vacA-derived probe 29 gctttagtrg ggytattggt 2030 19 DNA Artificial Sequence P1M1new vacA-derived probe 30 ttgatackggtaatggtgg 19 31 20 DNA Artificial Sequence P2M1new vacA-derived probe 31kggtaatggt ggtttcaaca 20 32 20 DNA Artificial Sequence P1M2newvacA-derived probe 32 kggtaatggt ggtttcaaca 20 33 21 DNA ArtificialSequence P2M2new vacA-derived probe 33 agagcgataa yggkctaaac a 21 34 21DNA Artificial Sequence P1M3 vacA-derived probe 34 agggtagaaa tggtatcgaca 21 35 23 DNA Artificial Sequence HpdiaS1 vacA-derived probe 35catgcygcct tctttacaac cgt 23 36 23 DNA Artificial Sequence HpdiaS2vacA-derived probe 36 catgccgcct ttttcacrac cgt 23 37 23 DNA ArtificialSequence HpdiaS3 vacA-derived probe 37 catgccgctc tttttacaac cgt 23 3823 DNA Artificial Sequence HpdiaS4 vacA-derived probe 38 catgccgccttttttacaac cgt 23 39 23 DNA Artificial Sequence HpdiaS5 vacA-derivedprobe 39 agtcgcgcyt ttttyacaac cgt 23 40 184 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 40 ccctctggtt tctctcgctttagtaggagc attggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccgtgatcattcc agccattgtt gggggtatcg ctacaggcac 120 cgctgtagga acggtctcagggcttcttag ttggggacta aaacaagccg aagaagccaa 180 taaa 184 41 199 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 41tcgccctctn gtttctctcg ctttagtagn agcattngtc agcatcacac cgcaacanag 60tcatgccgcc tttttcacaa ccgtnatcat tccagccatt gttgggggta tngctacagg 120caccgctgta ggaacggtct cagggcttct tagttgggga ctaaaacaag ccgaagaagc 180caataaaacc ccagataaa 199 42 227 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 42 gaatcgccct ytggtttctc ttgctttagtaggagcattg rttagyryca yaccgcaaca 60 aagtcatgcc gcctttttya craccgtgatcattccagcc attgttggrg gtatcgctac 120 aggcactgct gtaggaacgg tctcagggcttcttagttgg ggrctcaaac aagccgaaga 180 agcsaataaa accccrgata aacccgataaagtttggcgc attcaag 227 43 176 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 43 gccctttagt ttctctcgct ttagtggggttattggtcag catcacacca caaaaaagtc 60 atgccgcctt tttcacaacc gtgatcattccagccattgt tggaggtatc gctacaggtg 120 ctgctgtagg aacggtctca gggcttcttggttgggggct caaacaagcc gaagaa 176 44 185 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 44 gccctctggt ttctctcgctttagtaggag cattggtcag catcacaccg caacaaagtc 60 atgccgcctt tttcacaaccgtgatcattc cagccattgt tggaggtatc gctacaggcg 120 ctgctgtagg aacggtctcagggcttctta gctgggggct caaacaagcc gaagaagcca 180 ataaa 185 45 204 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 45aatcgccctc tggtttctct cgctttagta ggagcattgg tcagcatcac accgcaacaa 60agtcatgccg ccttttttac aaccgtgatc attccagcca ttgttggagg tatcgctaca 120ggcgctgctg taggaacggt ctcagggctt cttagctggg ggctcaaaca agccgaacaa 180gccaataaag ccccggacaa accc 204 46 207 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 46 gaatcgccct ttagtttctcttgctttagt aggagcattg gtcagcatca caccgcaaca 60 aagtcatgcc gcctttttcacaaccgtgat cattccagcc attgttgggg gtatcgctac 120 aggcgctgct gtaggaacggtttcagggct tcttggctgg gggctaaaac aagccgaaga 180 agccaataaa accccagataaacccga 207 47 207 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 47 caatcgccct ctagtttctc tcgctttagt aggagcattggtcagcatca caccgcaaca 60 aagtcatgcc gcctttttca caaccgtgat cattccagccattgtggggg gtatcgctac 120 aggcgctgct gtaggaacgg tctcagggct tcttagctgggggctcaaac aagccgaaga 180 agccaataaa accccggaca aacccga 207 48 207 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 48caatcgccct ctggtttctc ttgctttagt aggagcgtta gtcagcatca caccgcaaca 60aagtcatgcc gcctttttca caaccgtgat cattccagcc attgttgggg ggatcgctac 120aggcgctgct gtaggaacgg tctcagggct tcttagctgg gggctcaaac aagccgaaga 180agccaataaa accccagata aacccga 207 49 176 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 49 ccctttagtt tctcttgttttagcaggagc gttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacgaccgtgatcattcc agccattgtt gggggtatcg ctacaggcac 120 cgctgtagga acggtttcagggcttcttag ctgggggctc aaacaagccg aagaag 176 50 187 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 50 ccctttagtttctcctgttt tagcaggagc gttgattagc tccataccgc aacaaagtca 60 tgccgcctttttcacaaccg tgatcattcc agccattgtt gggggtatcg ctacaggcac 120 tgctgtaggaacggtctcag ggcttcttag ctgggggctc aaacaagcyg aasaagcsaa 180 taaagcc 18751 193 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 51 cttgagtttc tcttgtttta gcaggagcgt tgattagcgc cataccgcaacaaagtcatg 60 ccgccttttt cacgaccgtg atcattccag ccattgttgg gggtatcgctacaggcaccg 120 ctgtaggaac ggtttcaggg cttcttagct gggggctcaa acaagccgaagaagccaata 180 aaaccccaga taa 193 52 196 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 52 gccctttagt ttctctcgttttagcaggag cgttgattag ctccataccg caacaaagtc 60 atgccgcctt tttcacaaccgtgatcattc cagccattgt tgggggtatc gctacaggca 120 ccgctgtagg aacggtttcagggcttctta gctgggggct caaacaagcc gaacaagcca 180 ataaagcccc ggacaa 196 53131 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 53 aatcgccctt tagtttctct tgttttagca ggagcgttga ttagcgccataccgcaacaa 60 agtcatgccg cctttttcac gaccgtnatc attccagcca ttgttnnnngtatcgctaca 120 ggcaccgctg t 131 54 185 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 54 ctacgccctt tagtttctctcgttttagca ggagcgttga ttagcgccat accgcaacaa 60 agtcatgccg cctttttcacaaccgtgatc attccagcca ttgttggggg catcgcttca 120 ggcgctgctg taggaacggtctcagggctt cttagttggg gactcaaaca agccgaagaa 180 gcgaa 185 55 201 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 55aatcgccctt tagtttctct cgttttagca ggagcgttga ttagcgccat accgcaagag 60agtcatgccg cctttttcac aaccgtnatc attccagcca ttgttggggg tatcgctaca 120ggcaccgctg taggaacggt ctnagggctt yttagttggg gactnwaaca agccgaagaa 180gccaataaaa ccccggataa a 201 56 187 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 56 ccctttagtt tctctcgttt tagcannagcgttgattagc rccataccgc aagagagtca 60 tgccgccttt tttacaaccg tnatcattccagccattgtt gggggtatcg ctacaggygc 120 tgctgtagga acggtctcag ggcttcttagctgggggctc aaacaagccg aacaagccaa 180 taaagcc 187 57 187 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 57 ccctttggtttctctcgttt tagcaggagc gttgattagc tccataccgc aagagagtca 60 tgccgcctttttcacaaccg tgatcattcc agccattgtt gggggtatcg ctacaggcgc 120 tgctgtaggaacggtctcag ggcttcttag ctgggggctc aaacaagccg aagaagcgaa 180 taaaacc 18758 187 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 58 ccctttagtt tctctcgttt tagcaggagc gttgattagc tccataccgcaagagagtca 60 tgccgccttt ttcacaaccg tgatcattcc agccattgtt gggggtatcgctacaggcac 120 cgctgtagga acggtctcag ggcttcttag ctggggactc aaacaagccgaagaagcgaa 180 taaaacc 187 59 185 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 59 ccctttagtt tctctcgttt tagcagnagcgttnattagt gccataccgc aananagtca 60 tgccgccttt ttcacnaccg tnatcattccagccattgtt gggggtatcg ccacaggcac 120 cgctgtagga acggtctcag ggcttcttagttggggactn aaacaagccg aagaagcgaa 180 taaaa 185 60 199 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 60 caggagcgttgattagtgcc ataccgcaag agagtcatgc cgcctttttc acgaccgtga 60 tcattccagccattgttggg ggtatcgcca caggcaccgc tgtaggaacg gtctcagggc 120 ttcttagttggggactcaaa caagccgaag aagcgaataa aaccccagta taaacccgat 180 aaagtttggcgcattcaag 199 61 188 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 61 tttctctcgt tttagcagga gcgttgatta gctccataccgcaagagagt catgccgcct 60 ttttcacaac cgtgatcatt ccagccattg ttgggggtatcgctacaggc gctgctgtag 120 gaacggtctc agggcttctt agctgggggc tcaaacaagccgaagaagcg aataaaaccc 180 cagataaa 188 62 206 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 62 gaatcgccct ttagtttctctcgttttagc aggagcgttg attagtgcca taccgcaaga 60 gagtcatgcc gcctttttcacgaccgtgat cattccagcc attgttgggg gtatcgccac 120 aggcaccgct gtaggaacggtctcagggct tcttagttgg ggactcaaac aagccgaaga 180 agcgaataaa accccagtataaaccc 206 63 206 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 63 aatcgccctt tggtttctct cgttttagca ggagcgttgattagctccat accgcaagag 60 agtcatgccg cctttttcac aaccgtgmtc attccagccattgttggggg tatggctaca 120 ggcgctgctg taggaacggt ctnagggctt yttagctgggggctnwaaca agccgaagaa 180 gcgaataaaa ccccagataa acccga 206 64 201 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 64aatcgcccta ttatttctct cgttttagca ggagcgttga ttagctccat accgcaagaa 60agtcatgccg cctttttcac gaccgtgatc attccagcca ttgttggggg tatcgccaca 120ggcgctgctg taggaacggt ctcagggctt cttagctggg ggctcaaaca agccgaacaa 180gccaataaag ccccggacaa a 201 65 197 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 65 gccctttagt ttctcttgtt ttagcaggagcgttgattag tgccataccg caagagagct 60 atgccgcctt tttcacaacc gtgatcattccagccattgt tgggggtatc gctacaggca 120 ccgctgtagg aacggtctca gggcttcttagttggggact caaacaagcc gaagaagcga 180 ataaaacccc agataaa 197 66 201 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 66aatcgccctt tagtttctct cgttttagca ggagcgttta ttagcgccat accgcaagag 60agtcatgccg cctttttcac gaccgtgatc attccagcca ttgttggggg tatcgccaca 120ggcaccgctg taggaacggt ctcagggctt cttagttggg gactcaaaca agccgaagaa 180gcgaataaaa ccccagataa a 201 67 195 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 67 gtgggggtgt taatgggcac cgaactaggggctaatacgc caaacgatcc catacacagc 60 gagagtcgcg cctttttcac aaccgtgatcattccagcca ttgttggggg tatcgccaca 120 ggcactgctg taggaacggt ctcagggcttcttagttggg gactcaaaca agccgaagaa 180 gcgaataaaa cccca 195 68 196 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 68agtgggggtg ttaatgggca ctgaactagg ggctaacacg ccaaacgatc ccatacacag 60cgagagtcgc gcctttttta caaccgtgat cattccagcc attgttgggg gtatcgctac 120aggcgctgct gtaggaacgg tttcagggct tcttagctgg gggctcaaac aagccgaaca 180agccaataaa gccccg 196 69 196 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 69 tagtgggggc attaatgagt accgaactaggggctaacac gccaaatgat cccatacaca 60 gcgagagtcg cgcctttttt acaaccgtgatcattccagc cattgttggg ggtatcgcta 120 caggcgctgc tgtaggaacg gtctcagggcttcttagtcg ggggctcaaa caagccgaac 180 aagccaataa agcccc 196 70 232 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 70caatcgccct attatctctc tcgctctagt gggggtgtta atgggtaccg aactaggggc 60taacacgcca aacgatccca tacacagcga gagtcgcgcc ttttttacaa ccgtgatcat 120tccagccatt gttgggggta tcgctacagg cgctgctgta ggaacggttt cagggcttct 180tagctggggg ctcaaacaag ccgaacaagc caataaagcc ccggacaaac cc 232 71 228 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 71aatcgcccta ttatctctct cgctctagtg ggggtgttaa tgggtaccga actaggggct 60aacacgccaa acgatcccat acacagcgag agtcgcgcct ttttcacaac cgtgatcatt 120ccagccattg ttggaggtat cgctacaggt gctgctgtag gaacggtctc agggcttctt 180agctgggggc tcaaacaagc cgaacaagcc aataaagccc cggacaaa 228 72 228 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 72aatcgcccta ttatctctct cgctttagtg ggggtrttaa tgggcaccga actaggggct 60aacacgccaa acgatcccat acacagcgag agtcgcgcct ttttcacaac cgtgatcatt 120ccagccattg ttgggggtat cgctacaggc gctgctgtag gaacggtctc agggcttctt 180agctgggggc tcaaacaagc cgaacaagcc aataaagccc cggataaa 228 73 233 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 73aatcgcccta ttatctctct cgctttagag ggggtgttaa taggcaccga actaggggct 60aacacgccaa atgatcccat acacagcgag agtcgcgcct tttttacaac cgttattatt 120ccagccattg ttgggggtat cgctacaggc gctgctgtag gaacggtctc agggcttctt 180agctgggggc tcaaacaagc cgaacaagcc aataaagccc cggataaacc cga 233 74 300DNA Artificial Sequence Helicobacter pylori vacA nucleic acid sequence74 tttaaaggtg gatgctcata cagctaattt taaaggtatt gatacgggta atggtggttt 60caacacctta gattttagtg gtgttacagg taaggtcaat atcaacaagc tcatcacagc 120ttccactaat gtggccgtta aaaacttcaa cattaatgaa ttgattgtta aaaccaatgg 180tgtgagtgtg ggggaataca ctcattttag cgaagatata ggcagtcaat cgcgcatcaa 240taccgtgcgt ttggaaactg gcactaggtc aatcttttct gggggtgtta aatttaaagg 300 75300 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 75 tttaaaggtg gatgctcata cagctaattt taaaggtatt gatacgggtaatggtggttt 60 caacacctta gattttagtg gtgttacagg taaggtcaat atcaacaagctcattacggc 120 ttccactaat gtggccgtta aaaacttcaa cattaatgaa ttgttggttaagaccaatgg 180 ggtgagtgtg ggggaataca ctcattttag cgaagatata ggcagtcaatcgcgcatcaa 240 taccgtgcgt ttggaaactg gcactaggtc aatcttttct gggggtgtcaaatttaaagg 300 76 300 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 76 tttaaaagtg gatgctcata cagctaattt taaaggtattgatacgggta atggtggttt 60 caacaccttg gattttagtg gcgttacaga caaagtcaatatcaacaagc tcatcacagc 120 ttccactaat gtggccatta aaaacttcaa cattaatgaattgttggtta agaccaatgg 180 ggtgagtgtg ggggaataca ctcattttag cgaagatataggcagtcaat cgcgcatcaa 240 caccgtgcgt ttagaaactg gcactaggtc aatcttttctgggggtgtca aatttaaaag 300 77 300 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 77 tttaaaggtg gatgctcata cagctaattttaaaggtatt gatacgggta atggtggttt 60 caacacctta gattttagtg gtgttacaggtaaggtcaat atcaacaagc tcatcacagc 120 ttccactaat gtggccgcta aaaacttcaacattaatgaa ttgattgtta aaaccaatgg 180 ggtgagtgtg ggggaataca ctcattttagcgaagatata ggcagtcaat cgcgcatcaa 240 taccgtgcgt ttggaaactg gcactaggtcaatctattct ggcggtgtta aatttaaagg 300 78 300 DNA Artificial SequenceHelicobacter pylori vacA nucleic acid sequence 78 tttaaaagtg gatgctcatacagctaattt taaaggtatt gatacgggta atggtggttt 60 caacacctta gattttagtggtgttacagg taaggtcaat atcaacaagc tcatcacagc 120 ttccactaat gtggccgctaaaaacttcaa cattaatgaa ttgattgtta aaaccaatgg 180 ggtgagtgtg ggggaatacactcattttag cgaagatata ggcagtcaat cgcgcatcaa 240 taccgtgcgt ttggaaactggcactaggtc aatctattct ggcggtgtta aatttaaagg 300 79 300 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 79 tttaaaagtggatgctcata cagctaattt taaaggtatt gatactggta atggtggttt 60 caacaccttagattttagtg gtgttacaaa caaagtcaat atcaacaagc tcattacagc 120 ttccactaatgtggccgtta aaaacttcaa cattaatgaa ttgttggtta agattaatgg 180 ggtgagtgtgggggaataca cttattttag cgaagatata ggcagtcaat cgcgcatcaa 240 caccgtgcgtttggaaactg gcactaggtc aatctattct ggcggtgtta aatttaaagg 300 80 300 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 80tttaaaggtg gatgctcata cagctaattt taaaggtatt gatacgggta atggtggttt 60caacacctta gattttagtg gtgttacagg taaggtcaat atcaacaagc tcatcacggc 120ttccactaat gtggccgtta aaaacaacaa cattaatgaa ttggtggtta aaaccaatgg 180gataagtgtg ggggaataca ctcattttag cgaagatata ggcagtcaat cgcgcatcaa 240taccgtgcgt ttggaaacag gcactaggtc aatcttttct gggggtgtca aatttaaaag 300 81300 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 81 tttaaaagtg gatgctcata cagctaattt taaaggtatt gatacgggtaatggtggttt 60 caacacctta gattttagtg gtgttacagg taaggtcaat atcaacaagctcattacggc 120 ttccactaat gtagccgtta aaaacttcaa cattaatgaa ttgttggttaagaccaatgg 180 ggtgagtgtg ggggaataca ctcattttag cgaagatata ggcagtcaatcgcgcatcaa 240 caccgtgcgt ttggaaactg gcactaggtc aatcttttct gggggtgtcaaatttaaaag 300 82 300 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 82 tttaaaggtg gatgctcata cagctaattt taaaggtattgatacgggta atggtggttt 60 caacacctta gattttagtg gtgttacagg taaggtcaatatcaacaagc tcatcacagc 120 ttccactaat gtggccgtta aaaacttcaa cattaatgaattgattgtta aaaccaatgg 180 gataagtgtg ggggaataca ctcattttag cgaagatataggaagtcaat cgcgcatcaa 240 taccgtgcgt ttggaaactg gcactagatc aatcttttctgggggtgtta aatttaaagg 300 83 375 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 83 tttaagagtg gacgctcata cagcttattttaatggcaat atttatctgg gaaaatccac 60 gaatttaaga gtgaatggcc atagcgctcattttaaaaat attgatgcca gcaagagcga 120 taacgggcta aacactagcg ctttggatttcagcggcgtt acagacaaag tcaatatcaa 180 caagctcact acatctgcca ctaatgtgaacgttaaaaac tttgacgtta aggaattggt 240 ggttacaacc cgtgttcaga gttttgggcaatacactatt tttggcgaaa atataggcga 300 taagtctcgc attggtgtcg tgagtttgcaaacgggatat agcccggcct attctggggg 360 cgttactttt aaaag 375 84 375 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 84cttaagagtg gatgctcata cagcttattt taatggcaat atttatttgg gaaaatccac 60gaatttaaga gtgaatggcc atagcgctca ttttaaaaat attgatgcca gtaagagcga 120taacgggcta aacactagtg ctttggattt tagcggcgtt acagataaag tcaatatcaa 180caagctcact acatctgcca ctaatgtgaa cgttaaaaac tttgacatta aggaattggt 240ggttacaacc cgagttcaaa gttttgggca atacactatt tttggcgaaa atataggcga 300taagtctcgc attggtgtcg ttagtttgca aacgggatat agcccggcct attctggggg 360cgttactttt aaaag 375 85 374 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 85 tttaagagtg gatgctcata cagcttattttaatggcaat atttatctgg gaaaatccac 60 gaatttaaga gtgaatggcc atagcgctcattttaaaaat attgatgcca gtaagagcga 120 taacgggcta aacactacca ctttggatttcagcggcgtt acagataaag tcaatatcaa 180 caagctcact acatctgcca ctaatgtgaacattaaaaac tttgacatta aggaattagt 240 ggttacaacc cgagttcaga gttttgggcaatacactatt tttggcgaaa atataggcga 300 taagctgcac attggtgtcg tgagtttgcaaacgggatat agcccagcct attctggggg 360 gcttactttt aaag 374 86 375 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 86tttaagagtg gatgctcata cagcttattt taatggcaat atttatctgg gaaaatccac 60gaatttaaga gtgaatggcc atagcgctca ttttaaaaat attgatgcca gtaagagcga 120taacgggcta aacactagct ctttggattt cagtggcgtt acagacaaag tcaatatcaa 180caagctcact acatctgcca ctaatgtgaa cgttaaaaac tttgacatta aggaattggt 240ggttacaacc cgcgttcaga gttttgggca atacactatt tttggcgaaa atataggcga 300taagtctcgc attggtgtcg ttagtttgca aacgggatat agcccggcct attctggggg 360cgttactttt aaaag 375 87 365 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 87 gatgctcata cagcttattt taatggcaatatttatctgg gaaaatccac gaatttaaga 60 gtgaatggcc atagcgctca ttttaaaaatattgatgcca gtaagagcga taacgggcta 120 aacactagcg ctttggattt yagcggcgttacagayaaag tcaatatcaa caagctcact 180 acatctgcca ctaatgtgaa cgttaaaaactttgacatta aggaattagt ggttacaacc 240 cgagttcaaa gttttgggca atacactatttttggcgaaa atataggcga taagtctcgc 300 attggtgtcg ttagtttgca aacgggatatagcccggcct attctggggg cgttactttt 360 aaaag 365 88 375 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 88 tttaagaggggatgctcata cagcttattt taatggcaat atttatttgg gaaaatccac 60 gaatttaagagtgaatggcc atagcgctca ttttaaaaat attgatgcca gtaagagcga 120 taacgggctaaacactagcg ytttggattt tagcggcgtt acagayaaag tcaatatcaa 180 caagctcactacatctgcca ctaatgtgaa crttaaaaac tttgayatta aggaattggt 240 ggttacaacccgagttcaaa gttttgggca atacactatt tttggcgaaa atataggcga 300 tmagtctcgcattggtgtcg ttagtttgca aacgggatat agcccrgcct attctggggg 360 cgttacttttaaaag 375 89 375 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 89 tttaagcgtg gatgctcata cagcttattt taatggtaatatttatctgg gaaaatccac 60 gaatttaaga gtgaatggcc atagcgctca ttttaaaaatattgatgcca caaagagcga 120 taacgggcta aacactagcg ctttggattt cagcggcgttacagataaag tcaatatcaa 180 caagctcact acatctgcca ctaacgtgaa cattaaaaactttgacatta aggaattggt 240 ggttacaacc cgagttcaaa gttttgggca atacactatttttggcgaaa atataggcga 300 taagtctcgc attggtgtcg tgagtttgca aacgggatatagcccggcct attctggggg 360 cgttactttt aaaag 375 90 375 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 90 tttaagagtggatgctcata cagcttattt taatggcaat atttatctgg gaaaatccac 60 gaatttaaaagtgaatggcc atagcgctca ttttaaaaat attgatgcca gtaagagcga 120 taatggtctaaacactagtg ctttggattt gagcggcgtt acagacaaag tcaatatcaa 180 caagctcactacagctgcca ctaatgtgaa cattaaaaac tttgacatta aggaattagt 240 ggttacgacccgtgttcaga gttttgggca atacactatt tttggcgaaa atataggaga 300 tcaatcgcgcattggtgtcg ttagtttgca aactggctat agcccggcct attctggggg 360 cgttacttttaaaag 375 91 375 DNA Artificial Sequence Helicobacter pylori vacAnucleic acid sequence 91 cttaagagtg gatgctcata cagcttattt taatggcaatatttatctgg gaaaatccac 60 gaatttaaga gtgaatggcc atagcgctca ttttaaaaatattgatgcta gtaagagcga 120 taacgggcta aacactagcg ctttggattt tagcggcgttacagacaaag tcaatatcaa 180 caagctcact acatctgcca ctaatgtgaa cattaaaaactttgacatta aggaattggt 240 ggttacaacc cgagttcaaa gttttgggca atacactatttttggcgaaa atataggcga 300 taagtctcgc attggtgtcg tgagtttgca aacgggatatagcccggcct attctggggg 360 cgttactttt aaaag 375 92 449 DNA ArtificialSequence Helicobacter pylori cagA nucleic acid sequence 92 atgactaacgaaaccattaa ccaacaacca caaagcgaag cggcttttaa cccgcagcaa 60 tttatcaataatcttcaagt agcttttctt aaagttgata acgctgtcgc ttcatacgat 120 cctgatcaaaaaccaatcgt tgataagaac gatagggata ataggcaagc ttttgatgga 180 atctcgcaattaagggaaga atactccaat aaagcgatca aaaatcctac caaaaagaat 240 cagtatttttcagactttat caataagagc aatgatttaa tcaacaaaga cgctctcatt 300 gatgtagaatcttccacaaa gagctttcag aaatttgggg atcagcgtta ccgaattttc 360 acaagttgggtgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat 420 tttatggaaaatatcataca accccctat 449 93 449 DNA Artificial Sequence Helicobacterpylori cagA nucleic acid sequence 93 atgactaacg aaaccattaa ccaacaaccacaaaccgaag cggcttttaa cccgcagcaa 60 tttatcaata atcttcaagt agcttttcttaaagttgata acgctgtcgc ttcatacgat 120 cctgatcaaa aaccaatcgt tgataagaacgatagggata acaggcaagc ttttgatgga 180 atctcgcaat taagggaaga atactccaataaagcgatca aaaatcctac caaaaagaat 240 cagtattttt cagactttat caataagagcaatgatttaa tcaacaaaga cgctctcatt 300 gatgtagaat cttccacaaa gagctttcagaaatttgggg atcagcgtta ccgaattttc 360 acaagttggg tgtcccatca aaacgatccgtctaaaatca acacccgatc gatccgaaat 420 tttatggaaa atatcataca accccctat 44994 449 DNA Artificial Sequence Helicobacter pylori cagA nucleic acidsequence 94 atggctaacg aaactattaa ccaacaacca caaaccgaag cggcttttaacccgcagcaa 60 tttatcaata atcttcaagt agcttttctt aaagttgata acgctgtcgcttcatacgat 120 cctgatcaaa aaccaatcgt tgataagaac gatagggata acaggcaagcttttgatgga 180 atctcgcaat taagggaaga atactccaat aaagcgatca aaaatcctaccaaaaagaat 240 cagtattttt cagactttat caataagagc aatgatttaa tcaacaaagacgctctcatt 300 gatgtagaat cttccacaaa gagctttcag aaatttgggg atcagcgttaccgaattttc 360 acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatcgatccgaaat 420 tttatggaaa atatcataca accccctat 449 95 449 DNA ArtificialSequence Helicobacter pylori cagA nucleic acid sequence 95 atgactaacgaaaccattaa ccaacaacca caaaccgaag cggcttttaa cccgcagcaa 60 tttatcaataatcttcaagt ggcttttctt aaagttgata acgctgtcgc ttcatacgat 120 cctgatcaaaaaccaatcgt tgataagaac gatagggata ataggcaagc ttttgatgga 180 atctcgcaattaagggaaga atactccaat aaagcgatca aaaatcctac caaaaagaat 240 cagtatttttcagactttat caataagagc aatgatttaa tcaacaaaga cgctctcatt 300 gatgtagaatcttccacaaa gagctttcag aaattttggg atcagcgtta ccgaattttc 360 acaagttgggtgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat 420 ttcatggaaaatatcataca accccctat 449 96 449 DNA Artificial Sequence Helicobacterpylori cagA nucleic acid sequence 96 atgactaacg aaaccattaa ccaacaaccacaaaccgaag cggcttttaa cccgcagcaa 60 tttatcaata atcttcaagt ggcttttcttaaagttgata acgctgtcgc ttcatacgat 120 cctgatcaaa aaccaatcgt tgataagaacgatagggata acaggcaagc ttttgatgga 180 atctcgcaat taagggaaga atactccaataaagcgatca aaaatcctac caaaaagaat 240 cagtattttt caaactttat caataagagcaatgatctaa tcaacaaaga caatctcatt 300 gatgtagaat cttccaaaaa gagctttcagaaatttgggg atcagcgtta ccgaattttc 360 acaagttggg tgtcccatca aaacgatccgtctaaaatca acacccgatc gatccgaaat 420 tttatggaaa atatcataca accccctat 44997 449 DNA Artificial Sequence Helicobacter pylori cagA nucleic acidsequence 97 atgactaacg aaaccattaa ccaacaacca caaaccgaag cggcttttaacccgcagcaa 60 tttatcaata atcttcaagt agcttttctt aaagttgata acgctgtcgcttcatacgat 120 cctgaccaaa aaccaatcgt tgataagaac gatagggata acaggcaagcttttgataga 180 atctcacaat taagggagga atactccaat aaagcgatca aaaatcctaccaaaaagaat 240 cagtattttt cagactttat cgataagagc aacgatttaa tcaacaaagacgctctcatt 300 gatgtagaat cttccacaaa gagctttcag aaatttgggg atcagcgttaccgaattttc 360 acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatcgatccgaaat 420 tttatggaaa atatcataca accccctat 449 98 449 DNA ArtificialSequence Helicobacter pylori cagA nucleic acid sequence 98 atgactaacgaaaccattaa ccaacaacca caaaccgaag cggcttttaa cccgcagcaa 60 tttatcaataatcttcaagt ggcttttctt aaagttgata acgctgtcgc ttcatacgat 120 cctgatcaaaaaccaattat tgataagaac gatagggata acaggcaagc ttttgatgga 180 atctcgcaattaagggaaga atattccaat aaagcgatca aaaatcctac caaaaagaat 240 cagtatttttcagactttat cgataagagc aatgatttaa tcaacaaaga caatctcatt 300 gatgtagaatcttccacaaa gagctttcag aaatttgggg atcagcgtta ccgaattttc 360 acaagttgggtgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat 420 tttatggaaaatatcataca accccctat 449 99 449 DNA Artificial Sequence Helicobacterpylori cagA nucleic acid sequence 99 atgactaacg aaaccattaa ccaacaaccacaaaccgaag cggcttttaa cccgcagcaa 60 tttatcaata atcttcaagt agcttttcttaaagttgata atgctgtcgc ttcatacgat 120 tctgatcaaa aaccaatcat tgataagaacgatagggata acaggcaagc ttttgataga 180 atctcgcaat taagggaaga atactccaataaagcgatca aaaatcctac caaaaagaat 240 cagtattttt cagactttat cgataagagcaacgatttaa tcaacaaaga caatctcatt 300 gatgtagaat cttccacaaa gagctttcagaaatttgggg atcagcgtta ccgaattttc 360 acaagttggg tgtcccatca aaatgatccgtctaaaatca acacccgatc gatccgaaat 420 tttatggaaa atatcataca accccctat 449100 449 DNA Artificial Sequence Helicobacter pylori cagA nucleic acidsequence 100 atgactaacg aaactattga ccaacaacca caaaccgaag cggcttttaacccgcagcaa 60 tttattaata atcttcaggt agcttttctt aagcttgata acgctgtcgcttcatttgat 120 cctgatcaaa aaccaatcgt tgataagaat gatagggata acaggcaagcttttgatgga 180 atctcgcaat taagggaaga atactccaat aaagcgatca aaaatcctaccaaaaagaat 240 cagtattttt cagactttat caataagagc aatgatttaa tcaacaaagacgctctcatt 300 gatgtagaat cttccacaaa gagctttcag aaatttgggg atcagcgttaccgaattttc 360 acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatcgatccaaaat 420 tttatggaaa atatcataca accccctat 449 101 449 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 101atgactaacg aaactattga ccaacaacca caaactgaag cggcttttaa cccgcagcaa 60tttatcaata atcttcaagt ggcttttctt aagcttgata acgctgtcgc ttcatttgat 120cctgatcaaa aaccaatcgt tgataagaac gatagggata acaggcaagc ttttgatgga 180atctcgcaat taagggaaga atactccaat aaagcgatca aaaatcctac caaaaagaat 240cagtattttt cagactttat caataagagc aatgatttaa tcaacaaaga cgctctcatt 300gatgtagaat cttccacaaa gagctttcag aaatttgggg atcagcgtta ccgaattttc 360acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat 420tttatggaaa atatcataca accccctat 449 102 449 DNA Artificial SequenceHelicobacter pylori cagA nucleic acid sequence 102 atgactaacg aaactattaaccaacagcca caaaccgaag cggcttttaa cccgcagcaa 60 tttatcaata atcttcaagtagcttttctt aagcttgata acgctgtcgc ttcatttgat 120 cctgatcaaa aaccaatcgttgataagaac gatagggata ataggcaggc ttttgatgga 180 atctcgcaat taagggaagaatactccaat aaagcgatca aaaatcctac caaaaagaat 240 cagtattttt cagactttatcaataagagc aatgatttaa tcaacaaaga caatctcatt 300 gatgtagaat cttccacaaagagctttcag aaatttgggg atcagcgtta ccgaattttc 360 acaagttggg tgtcccatcaaaacgatccg tctaaaatca acacccgatc gatccgaaat 420 tttatggaaa atatcatacaaccccctat 449 103 449 DNA Artificial Sequence Helicobacter pylori cagAnucleic acid sequence 103 atgactaacg aaaccattaa ccaacaacca caaaccgaagcggcttttaa cccgcagcaa 60 tttatcaata atcttcaagt ggcttttctt aagcttgataatgctgttgc ttcatttgat 120 cctgatcaaa aaccaatcgt tgataagaac gatagggataacaggcaagc ttttgatgga 180 atctcgcaat taagggaaga atactccaat aaagcgatcaaaaatcctac caaaaagaat 240 cagtattttt cagactttat cgataagagc aacgatttaatcaacaaaga caatctcatt 300 gatgtagaat cttccacaaa gagctttcag aaatttggggatcagcgtta ccgaattttc 360 acaagttggg tgtcccatca aaacgatccg tctaaaatcaacacccgatc gatccgaaat 420 tttatggaaa atatcataca accccctat 449 104 449DNA Artificial Sequence Helicobacter pylori cagA nucleic acid sequence104 atgactaacg aaaccattaa ccaacaacca caaaccgaag cggcttttaa cccgcagcaa 60tttatcaata atcttcaagt ggcttttctt aaagttgata acgctgtcgc ttcatacgat 120cctgatcaaa aaccaatcgt tgataagaac gatagggata ataggcaagc ttttgatgga 180atctcgcaat taagggaaga atactccaat aaagcgatca aaaatcctac caaaaagaat 240cagtattttt cagactttat caataagagc aatgatttaa tcaacaaaga cgctctcatt 300gatgtagaat cttccacaaa gagctttcag aaattttggg atcagcgtta ccgaattttc 360acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat 420ttcatggaaa atatcataca accccctat 449 105 449 DNA Artificial SequenceHelicobacter pylori cagA nucleic acid sequence 105 atgactaacg aaactattgatcaacaacca cgaaccgaag cggcttttaa cccgcagcaa 60 tttatcaata atcttcaagtagcttttctt aaagttgata acgttgtcgc ttcatttgat 120 cctaatcaaa aaccaatcgttgataagaac gatagggata acaggcaagc ttttgatgga 180 atctcgcaat taagggaagaatactccaat aaagcgatca aaaatcctgc caaaaagaat 240 cagtattttt cagactttatcaataagagc aatgatctaa tcaacaaaga caatctcatt 300 gatgtagaat cttccacaaagagctttcag aaatttgggg atcagcgtta ccaaattttc 360 acaagttggg tgtcccatcaaaacgatccg tctaaaatca acacccgatc gatccgaaat 420 tttatggaaa atatcatacaaccccctat 449 106 449 DNA Artificial Sequence Helicobacter pylori cagAnucleic acid sequence 106 atgactaacg aaaccattaa ccaacaacca caaaccgaagcggcttttaa cccgcagcaa 60 tttatcaata atcttcaagt ggcttttatt aaagttgataatgttgtcgc ttcatttgat 120 cctgatcaaa aaccaatcgt tgataagaat gatagggataataggcaagc ttttgagaaa 180 atctcgcagc taagggagga attcgctaat aaagcgatcaaaaatcctgc caaaaagaat 240 cagtattttt caagctttat cagtaagagc agtgatttaatcaacaaaga cagtctcatt 300 gatacaggtt cttccataaa gagctttcag aaatttgggactcagcgtta ccaaattttt 360 atgaattggg tgtcccatca aaaagatcca tctaaaatcaacacccaaaa aatccgaggt 420 tttatggaaa atatcataca accccctat 449 107 464DNA Artificial Sequence Helicobacter pylori cagA nucleic acid sequence107 atgactaacg aaactattga tcaaacaaga acaccagacc aaacacaaag ccaaacagct 60tttgatccgc aacaatttat caataatatt caagtggctt ttcttaaagt tgataacgct 120gtcgcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcagctaagg gaggaattcg ctaataaagc gatcaaaaat 240cctgccaaaa agaatcagta tttttcaagc tttatcagta agagcagtga tttagtcaac 300aaagacagtc tcattgatac aggttcttcc ataaagagct ttcagaaatt tgggactcag 360cgttaccaaa tttttatgaa ttgggtgtcc catcaaaaag atccatctaa aatcaacacc 420caaaaaatcc aagattttat ggaaaatatc atacaacccc ctat 464 108 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 108atgactaatg aaaccattga tcaaacaaca acaccagatc aaacaccaaa tcaaacagat 60tttgttccgc aacgatttat caataatctt caagtagctt ttattaaagt tgataacgct 120gtctcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaactaagg gaagaatacg ccaataaagc gatcaaaaat 240cctgccaaaa agaatcagta tttttcagac tttatcaata agagcaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttcc gtagagacct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgaa ttgggtgtcc cttcaaaaag atccgtctaa aatcaacacc 420cgacaaatcc gaaattttat ggaaaatatc atacaacccc ctat 464 109 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 109atgactaacg aaaccattga tcaaacaaca acaccagatc aaacaccaaa ccaaacggat 60tttgttccgc aacgatttat caataatctt caagtagctt tccttaaagt tgatagcgct 120gtcgcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaactaagg gaagaatacg ccaataaagc gatcaaaaat 240cctgccaaaa agaatcagta tttttcagac tttatcaata agagcaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttct gtagagagct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgag ttgggtgtcc cttcaaaaag atccgtctca aatcaacacc 420cgacaaatcc gaaattttat ggaaaatatc atacaacccc ctat 464 110 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 110atgactaacg aaaccattga tcaaacaaca acaccagatc aaacaccaag ccaaacagat 60tttgttccgc aacgatttat caataatctt caagtagctt ttcttaaagt tgataacgct 120gtcgcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaactaagg gaagaatacg ccaataaagc gatcaaaaat 240cctgccaaaa agaatcagta tttttcagac tttatcaata agaccaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttcc gtagatagct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgag ttgggtgtcc cttcaaaaag atccgtctaa aatcaacacc 420caacaaatcc gaaattttat ggaaaatatc atacaacccc ctat 464 111 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 111atgactaacg aaaccattga tcaaacaaca acaccagatc aaacaccaaa tcaaacagat 60tttgttccgc aacgatttat caataatctt caagtagctt ttattaaagt tgatgacgct 120gtcgcttcat ttgatcccga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaactaagg gaagaatacg ccaataaagc gatcaaaaat 240cccaccaaaa agaatcagta tttttcagac tttatcaata agaccaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttcc gtagagagct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgag ttgggtgtcc cttcaaaaag atccgtctaa aatcaacacc 420caacaaatcc gaaattttat ggaaaatatc atacaacccc ctat 464 112 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 112atgactaacg aaaccattga tcaaacaaca acaccagatc aaacaccaaa tcaaacagat 60tttgttccgc aacgatttat caataatctt caagtagctt ttattaaagt tgataacgct 120gttgcttcat ttgatcccga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaactaagg gaagaatacg ccaataaagc gatcaaaaat 240cctgccaaaa agaatcagta tttttcagac tttatcaata agagcaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttcc gtagatagct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgag ttgggtgtcc cttcaaaaag atccgtctca aatcaacacc 420caacaaatcc aaaattttat ggaaaatatc atacaacccc ctat 464 113 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 113atgactaacg aaaccattga tcaaacaaca acaccagatc aaacactaaa ccaaacggat 60tttgttccgc aacgatttat caataatctt caagtagctt ttattaaagt tgataacgct 120gtcgctttat ttgatcccga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaactaagg gaagaatacg ccaataaagc gatcaaaaat 240cccaccaaaa agaatcagta tttttcagac tttatcaata agagcaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttcc gtagatagct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgag ttgggtgtcc cttcaaaaag atccgtctca aatcaacacc 420cgacaaatcc gaaattttat ggaaaatatc atacaacccc ctat 464 114 464 DNAArtificial Sequence Helicobacter pylori cagA nucleic acid sequence 114atgactaacg aaaccattga tcaaacaata acaccagatc aaacaccaaa ccaaacggat 60tttgttccgc aacgatttat caataatctt caagtagctt ttatcaaagt tgataacgct 120gtcgcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180caagcttttg agaaaatctc gcaattaagg gaagaatacg ccaataaagc gatcaaaaat 240cctgccaaaa agaatcagta ttttttagac tttatcaata agagcaatga tttgatcaac 300aaagacaatc tcattgctgt agattcttcc gtagatagct ttaagaaatt tggggatcag 360cgttaccaaa tttttacgag ttgggtgtcc cttcaaaaag atccgtctaa aatcaacacc 420caacaaatcc gaaattttat ggaaaatatc atacaacccc ctat 464 115 132 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 115ccctattatc tctctcgctc tagtgggggt gttaatgggt accgaactag gggctaacac 60gccaaacgat cccatacaca gcgagagtcg cgcctttttt acaaccgtga tcattccagc 120cattgttggg gg 132 116 132 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 116 ccctattatc tctctcgctc tagtgggggtgttaatgggt accgaactag gggctaacac 60 gccaaacgat cccatacaca gcgagagtcgcgcctttttc acaaccgtga tcattccagc 120 cattgttgga gg 132 117 132 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 117ccctattatc tctctcgctc tagtgggggt gttaatgggc accgaactag gggctaatac 60gccaaacgat cccatacaca gcgagagtcg cgcctttttc acaaccgtga tcattccagc 120cattgttggg gg 132 118 132 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 118 ccctattatc tctctcgctc tagtgggggtgttaatgggc accgaactag gggctaatac 60 gccaaacgat cccatacaca gcgagagtcgcgcctttttc acaaccgtga tcattccagc 120 cattgttggg gg 132 119 132 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 119ccctattatc tctctcgctc tagtgggggt gttaatgggc actgaactag gggctaacac 60gccaaacgat cccatacaca gcgagagtcg cgcctttttt acaaccgtga tcattccagc 120cattgttggg gg 132 120 132 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 120 ccctattatc tctctcgctc tagtgggggtgttaatgggc accgaactag gggctaacac 60 gccaaatgat cccatacaca gcgagagtcgcgcctttttc acaacygtga tcattccagc 120 cattgttggg gg 132 121 132 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 121ccctattatc tctctcgctt tagtgggggt rttaatgggc accgaactag gggctaacac 60gccaaacgat cccatacaca gcgagagtcg cgcctttttc acaaccgtga tcattccagc 120cattgttggg gg 132 122 132 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 122 ccctattatt tctctcgctt tagtgggggtgttaatgggc accgaactag gggctaacac 60 gccaaacgat cccatacaca gcgagagtcgcgcctttttt acaaccgtga tcattccagc 120 cattgttggg gg 132 123 132 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 123ccctattatc tctctcgctc tagtgggggc attaatgagt accgaactag gggctaacac 60gccaaatgat cccatacaca gcgagagtcg cgcctttttt acaaccgtga tcattccagc 120cattgttggg gg 132 124 132 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 124 ccctattatc tctctcgctt tagtgggggtattaataggc accgaactag gggctaatac 60 gccaaatgat cccatacaca gcgagagtcgtgcttttttc acaaccgtga tcattccagc 120 cattgttggg gg 132 125 132 DNAArtificial Sequence Helicobacter pylori vacA nucleic acid sequence 125ccctattatc tctctcgctc tagtgggggt gttaataagc accgaactag gggctaacac 60gccaaatgat cccatacaca gcgagagtcg cgcctttttc acaacgggga tcattccagc 120cattgttggg gg 132 126 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 126 ccctttagtt tctctcgctt tagtggggttattggtcagc atcacaccac aaaaaagtca 60 tgctgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 127 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 127 ccctttagtt tctctcgctt tagtggggttattggtcagc atcacaccac aaaaaagtca 60 tgctgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 128 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 128 ccctttagtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgctgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 129 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 129 ccctctagtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 130 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 130 ccctctagtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccadccattgtt ggggg 105 131 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 131 ccctttagtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccggccattgtt ggggg 105 132 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 132 ccctttagtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 133 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 133 ccctttagtt tctctcgctt tagtggggctattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 134 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 134 ccctttagtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttt ttcacaaccg tgatcattccggccattgtt ggggg 105 135 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 135 ccctttagtt tctctcgctt tagtggggctattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgattatcccggccattgtt ggggg 105 136 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 136 ccctttagtt tctctcgctt tagtggggctattggtcagc atcacaccac aaaaaagtca 60 tgccgccttt tttacaaccg tgatcattccagccattgtt gggag 105 137 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 137 ccctttagtt tctctcgctt tagtggggctattggtcagc atcacaccac aaaaaagtca 60 tgccgccttt tttacaaccg tgatcattccagccattgtt ggagg 105 138 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 138 ccctctggta tctctcgctt tagtggggctattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgaatattccagcmattgtt ggggg 105 139 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 139 ccctctggtt tctctcgctt tagtggggctattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttt ttcacaaccg tgatcattccggccattgtt ggggg 105 140 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 140 ccctctggtt tctctcgctt tagtggggctattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagcmattgtt ggggg 105 141 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 141 ccctctggtt tctctcgctt tagtggggctattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagccattgtt ggggg 105 142 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 142 ccctctggtt tctctcgctt tagtggggctattggtcagc atcacaccac aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagchattgtt ggggg 105 143 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 143 ccctctggtt tctctcgctt tagtggggctattggtcagc atcacaccac aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagcyattgtt ggagg 105 144 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 144 ccctctggtt tctctcgctt tagtggggttattggtcagc atcacaccgc aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagccattgtt ggagg 105 145 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 145 ccctttagtt tctctcgctt tagtggggttattggttagc atcacaccgc aaaaaagtca 60 tgccgccttt ttcacaaccg tgattattccagccattggt tgggg 105 146 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 146 ccctctggtt tctctcgctt tagtggggctattggtcagc atcacaccac aaaaaagtca 60 tgccgccttc tttacaaccg tgatcattccagccatcgtt ccccc 105 147 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 147 ccctttagtt tctcctgttt tagcaggagcgttgattagc tccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 148 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 148 ccctttagtt tctctcgttt tagcaggagcgttgattagt gccataccgc aagaaagtca 60 tgccgccttt tttacaaccg taatcattccagctattgtt ggggg 105 149 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 149 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 150 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 150 ccctttagtt tctctcgttt tagcaggagcgtttattagc gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 151 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 151 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aagaaagtta 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 152 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 152 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 153 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 153 ccctattatt tctctcgttt tagcaggagcgttgattagc tccataccgc aagaaagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 154 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 154 ccctttagtt tctctcgttt tagcagnagcgttnattagt gccataccgc aananagtca 60 tgccgccttt ttcacnaccg tnatcattccagccattgtt ggggg 105 155 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 155 ccctttggtt tctctcgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtg ggggg 105 156 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 156 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 157 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 157 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 158 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 158 ccctttagtt tctctcgttt tagcaggagcgttgattagc tccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 159 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 159 ccctttagtt tctcttgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 160 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 160 ccctttagtt tctcttgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 161 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 161 ccctttggtt tctctcgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagcvattgtg gggag 105 162 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 162 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 163 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 163 ccctttagtt tctctcgttt tagcaggagcgttgattagt gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 164 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 164 ccctttagtt tctctcgttt tagcaggagcgttgattagt gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 165 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 165 ccctttagtt tctctcgttt tagcaggagcgttggttagt gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 166 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 166 ccctttagtt tctctcgttt tagcaggagcgttgattagt gccataccgc aagagagtca 60 tgccgccttt ttcacaaccg tgatcattccarccattgtt ggggg 105 167 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 167 ccctttagtt tctcttgttt tagcaggagcgttgattagc gccataccgc aagagagtca 60 tgccgccttt ttcacaaccg taatcattccagccattgtt ggggg 105 168 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 168 ccctttagtt tctcttgttt tagcaggagcgttgattagc gccataccgc aagagagtca 60 tgccgccttt ttcacgaccg taatcattccagccattgtt ggggg 105 169 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 169 ccctttagtt tctctcgttt tagcaggagcgttgattagc gccataccgc aagagagtca 60 tgccgccttt ttcacaaccg tnatcattccagccattgtt ggggg 105 170 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 170 ccctttggtt tctctcgttt tagcaggagcgttgattagc tccataccgc aagagagtca 60 tgccgccttt ttcacacccg tgatcattccagccattgtt ggggg 105 171 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 171 ccctttggtt tctctcgttt tagcaggagcgttgattagc tccataccgc aagagagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 172 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 172 ccctttggtt tctctcgttt tagcaggagcgttgattagc tccataccgc aagagagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 173 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 173 ccctttggtt tctctcgttt tagcaggagcgttgattagc tccataccgc aagagagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 174 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 174 ccctttagtt tctctcgttt tagcaggagcgttgattagc tccataccgc aagagagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 175 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 175 ccctttagtt tctctcgttt tagcannagcgttgattagc rccataccgc aagagagtca 60 tgccgccttt tttacaaccg tgatcattccagccattgtt ggggg 105 176 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 176 ccctttagtt tctcttgttt tagcaggagcgttgattagc gccataccgc aacaaagtca 60 tgccgccttt ttcacgaccg tnatcattccagccattgtt nnnng 105 177 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 177 ccctctggtt tctctggttt tagcaggagcgttgattagc atcacaccac aacaaagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 178 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 178 ccctctggtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 179 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 179 ccctctggtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 180 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 180 ccctctggtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggagg 105 181 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 181 ccctctggtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt tttacaaccg tgatcattccagccattgtt ggagg 105 182 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 182 ccctttggtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacgaccg tgatcattccagccattgtt ggggg 105 183 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 183 ccctctggtt tctcttgctt tagtaggagcgttagtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 184 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 184 ccctctagtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtg ggggg 105 185 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 185 ccctttagtt tctcttgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgatcattccagccattgtt ggggg 105 186 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 186 ccctctngtt tctctcgctt tagtagnagcattngtcagc atcacaccgc aacanagtca 60 tgccgccttt ttcacaaccg tnatcattccagccattgtt ggggg 105 187 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 187 ccctctggtt tctctcgctt tagtaggagcgttggtcagc atcacaccgc aacaaagtca 60 tgccgccttt attacaaccg tgatcattccagccattgtt ggggg 105 188 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 188 ccctctggtt tctcttgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt tttacaaccg tgattattccagccattgtg ggggg 105 189 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 189 ccctctggtt tctcttgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgctctt tttacaaccg tgattattccagccattgtg ggggg 105 190 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 190 ccctctggtt tctcttgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgctctt tttacaaccg tgattattccagccattgtg ggggg 105 191 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 191 ccctctggtt tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgctctt tttacaaccg tgattattccagccattgtg ggggg 105 192 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 192 ccctytggtt tctcttgctt tagtaggagcattgrttagy rycayaccgc aacaaagtca 60 tgccgccttt ttyacraccg tgatcattccagccattgtt ggrgg 105 193 105 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 193 ccctattatc tctctcgctt tagtaggagcattggtcagc atcacaccgc aacaaagtca 60 tgccgccttt ttcacaaccg tgttcattccagccattgtt ggggg 105 194 362 DNA Artificial Sequence Helicobacter pylorivacA nucleic acid sequence 194 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccacaaagag cgataacggg 120 ctaaacacta gcactttgga ttttagcggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agtggttacg 240 acccgtgttc agagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgttagttt gcaaactggctatagcccgg cctattctgg gggcgttact 360 tt 362 195 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 195 gtggatgctcatacagctta ttttaatggc aatatttatc tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ccacaaagag cgataacggg 120 ctaaacactagtgctttgga tttgagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacattaaa aactttgaca ttaaggaatt agtggttaca 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtcg 300 cacattggtgtcgttagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt 362 196362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 196 gtggatgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccacaaagagcgataacggg 120 ctaaacacta gcgctttgga tttgagcggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacgttaaa aactttgaca ttaaggaattggtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgataagtcg 300 cgcattggtg tcgttagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 197 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 197 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccacaaagag cgataacggg 120 ctaaacacta gcgctttgga tttgagcggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacgttaaaaactttgaca ttaaggaatt ggtggttaca 240 acccgagttc aaagttttgg gcaatacactatttttggcg aaaatatagg cgataagtcg 300 cgcattggtg tcgttagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 198 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 198 gtggatgctcatacagctta ttttaatggt aatatttatc tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ccacaaagag cgataacggg 120 ctaaacactagcgctttgga tttcagcggc gttacagata aagtcaatat caacaagctc 180 actacatctgccactaacgt gaacattaaa aactttgaca ttaaggaatt ggtggttaca 240 acccgagttcaaagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgtgagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt 362 199362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 199 gtggatgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagtaagagcgataacggg 120 ctaaacacta gcactttgga ttttagcggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacgttaaa aactttgaca ttaaggaattggtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgatcagtct 300 cgcattggtg tcgtgagttt gcaaacggga tatagcccgg cttattctgggggcgttact 360 tt 362 200 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 200 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ctagtaagag cgataacggg 120 ctaaacacta gcgctttgga ttttagcggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt ggtggttaca 240 acccgagttc aaagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgtgagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 201 2 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 201 wc 2 202 362DNA Artificial Sequence Helicobacter pylori vacA nucleic acid sequence202 gtggatgctc atacagctta ttttaatggc aatgtttatc tgggaaaatc cacgaattta 60agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagcaagag cgataacggg 120ctaaacacta gtgctttgga ttttagcggc gttacagaca aagtcaatat caacaagctc 180actacatctg ccactaatgt gaacattaaa aactttgaca ttaaggaatt agtggttaca 240acccgagttc aaagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300cgcattggtg tcgtgagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt362 203 362 DNA Artificial Sequence Helicobacter pylori vacA nucleicacid sequence 203 gtggatgctc atacagctta ttttaatggc aatgtttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagcaagagcgataacggg 120 ctaaacacta gtgctttgga ttttagcggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacattaaa aactttgaca ttaaggaattagtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgtgagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 204 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 204 gtggatgctc atacagctta ttttaatggcaatgtttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccagcaagag cgataacggg 120 ctaaacacta gtgctttgga ttttagcggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agtggttaca 240 acccgagttc aaagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgtgagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 205 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 205 gtggatgctcatacagctta ttttaatggc aatgtttatc tgggaaaatc cacgaattta 60 agagtgaatgcccatagcgc tcattttaaa aatattgatg ccagcaagag cgataacggg 120 ctaaacactagtgctttgga ttttagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacattaaa aactttgaca ttaaggaatt agtggttaca 240 acccgagttcaaagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgtgagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt 362 206362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 206 gtggatgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccacaaagagcgataacggg 120 ctaaacacta gcgctttgga ttttagtggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacattaaa aactttgaca ttaaggaattagtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccag cctattctgggggcgttact 360 tt 362 207 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 207 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccagtaagag cgataacggg 120 ctaaacacta gtgctttgga ttttagcggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agttgttaca 240 acccgagttc aaagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgttagttt gcaaacgggatatagcccag cctattctgg gggcgttact 360 tt 362 208 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 208 gtggatgctcatacagctta ttttaatggc aatatttatc tgggaaaatc cacgaattta 60 aaagtgaatggccatagcgc tcattttaaa aatattgatg ccagtaagag cgataatggt 120 ctaaacactagcgctttgga ttttagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacattaaa aactttgaca ttaaggaatt agtggttaca 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgttagttt gcaaatggga tatagcccgg cctattctgg gggcgttact 360 tt 362 209362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 209 gtggatgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 aaagtgaatg gccatagcgc tcattttaaa aatattgatg ccagtaagagcgataacggg 120 ctaaacacta gcgctttgga tttgagcggc gttacagaca aagtcaatatcaacaagctc 180 actacagctg ccactaatgt gaacattaaa aactttgaca ttaaggaattggttgttacg 240 acccgtgttc agagttttgg acaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccgg cttattctgggggcgttact 360 tt 362 210 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 210 gtggatgctc atacagctaa ctttaatggcaatatttatt tgggaaaatc cacgaattta 60 agggtgaatg gccatagcgc tcattttaaaaatattgatg ccagtaagag cgataacggg 120 ctaaacacta gctctttgga tttcagtggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacgttaaaaactttgaca ttaaggaatt ggtggttaca 240 acccgagttc agagttttgg gcaatacactatttttggcg aaattatagg cgataagtct 300 cgcattggtg tcgttagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 211 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 211 gtggatgctcatacagctta ttttaatggc aatatttatt tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ccagtaagag cgataacggg 120 ctaaacactagtgctttgga ttttagcggc gttacagata aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacgttaaa aactttgaca ttaaggaatt ggtggttaca 240 acccgagttcaaagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgttagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt 362 212362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 212 gtggatgccc atacggctaa ctttaatggc aatatttatc tgggaaaatccacgaattta 60 aaagtgaatg gccatagcgc tcattttaaa aatattgatg ccacaaagagcgataacggg 120 ctaaacacta gcgctttgga tttgagtggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacattaaa aactttgaca ttaaggaattagtggttaca 240 acccgtgttc agagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 213 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 213 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 aaagtgaatg gccatagcgc tcattttaaaaatattgatg ccagtaagag cgataatggt 120 ctaaacacta gtgctttgga tttgagcggcgttacagaca aagtcaatat caacaagctc 180 actacagctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agtggttacg 240 acccgtgttc agagttttgg gcaatacactatttttggcg aaaatatagg agatcaatcg 300 cgcattggtg tcgttagttt gcaaactggctatagcccgg cctattctgg gggcgttact 360 tt 362 214 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 214 gtggatgctcatacagctta ttttaatggc aatatttatc tgggaaaatc cacgaattta 60 aaagtgaatggccatagcgc tcattttaaa aatattgatg ccactaagag cgataatggt 120 ctaaacactagcgctttgga tttgagcggc gttacaaaca aggtcaatat caacaagctc 180 actacagctgccactaatgt gtccattaaa aactttgaca ttaaggaatt agtggttacg 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgatcaatcg 300 cgcattggtgtcgttagttt gcaaactggc tatagcccgg cctattctgg gggcgttact 360 tt 362 215362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 215 gtggatgctc atacggctaa ctttaatggc aatgtttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagtaagagcgataacggg 120 ctaaacacta gcgctttgga ttttagcggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacattaaa aactttgaca ttaaggaattggtggttaca 240 acccgtgttc agagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 216 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 216 gtggatgctc atacagctta ttttaatggcaatgtttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccagcaagag cgataacggg 120 ctaaacacta gcgctttgga ttttagcggcgttacagata aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agtggttaca 240 acccgtgttc agagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgtgagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 217 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 217 gtggatgctcatacagctta ttttaatggc aatgtttatc tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ctagtaagag cgataacggg 120 ctaaacactagcgctttgga ttttagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacgttaaa aactttgaca ttaaggaatt agtggttaca 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgtgagttt gcaaacggga tatagcccag cctattctgg gggcgttact 360 tt 362 218362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 218 gtggacgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagcaagagcgataacggg 120 ctaaacacta gcgctttgga tttcagcggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacgttaaa aactttgacg ttaaggaattggtggttaca 240 acccgtgttc agagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgtgagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 219 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 219 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccagcaagag cgataacggg 120 ctaaacacta gcactttgga ttttagcggcgttacagata aagtcaatat caacaagctc 180 actacagctg ccactaatgt gaacgttaaaaactttgaca ttaaggaatt agtggttaca 240 acccgagttc aaagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgttagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 220 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 220 gtggatgctcatacagctta ttttaatggc aatatttatt tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ccagtaagag cgataacggg 120 ctaaacactagcgctttgga ttttagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacgttaaa aactttgaca ttaaggaatt agtggttaca 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgttagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt 362 221362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 221 gtggatgctc atacagctta ttttaatggc aatatttatt tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagtaagagcgataacggg 120 ctaaacacta gtgctttgga ttttagcggc gttacagata aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacgttaaa aactttgaca ttaaggaattggtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 222 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 222 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agattgaatg gccatagcgc tcattttaaaaatattgatg ccagtaagag cgataacggg 120 ctaaacacta gcgctttgga ttttagcggcgttacagaca aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacgttaaaaactttgaca ttaaggaatt ggtggttaca 240 acccgagttc agagttttgg gcaatactctatttttggcg aaaatatagg cgataagtcg 300 cgcattggtg tcgttagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 223 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 223 gtggacgctcatacagctta ttttaatggc aatatttatt tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ccacaaagag cgataacggg 120 ctaaacactagcactttgga tttgagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacattaaa aactttgaca ttaaggaatt agtggttacg 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgtgagttt gcaaacggga tatagcccgg cctattctgg gggcgttact 360 tt 362 224362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 224 gtggatgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccacaaagagcgataacggg 120 ctaaacatta gcactttgga ttttagcggc gttacagaca aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacattaaa aactttgaca ttaaggaattagtggttaca 240 acccgtgttc agagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 225 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 225 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaatttg 60 agagtgaatg gccataacgc tcattttaaaaatattgatg ccagtaagag cgataacggg 120 ctaaacacta gcactttgga tttgagcggcgttacagaca aagtcaatat caacaagctc 180 actacagctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agtggttacg 240 acccgtgttc agagttttgg gcaatacactatttttggcg aaaatatagg tgataagtct 300 cgcattggtg tcgttagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 226 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 226 gtggatgctcatacagctta ttttaatggc aatgtttatt tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcattttaaa aatattgatg ccagtgagag cgataacggg 120 ctaaacactagcgctttgga ttttagcggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacgttaaa aactttgaca ttaaggaatt ggtggttacg 240 acccgtgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtct 300 cgcattggtgtcgttagttt gcaaacggga tgtcgcccgg cctgttctgg gggcgttact 360 tt 362 227362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 227 ggggatgctc atacagctta ttttaatggc aatatttatt tgggaaaatccacgaattta 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagtaagagcgataacggg 120 ctaaacacta gcgytttgga ttttagcggc gttacagaya aagtcaatatcaacaagctc 180 actacatctg ccactaatgt gaacrttaaa aactttgaya ttaaggaattggtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgatmagtct 300 cgcattggtg tcgttagttt gcaaacggga tatagcccrg cctattctgggggcgttact 360 tt 362 228 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 228 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccacaaagag cgataatggt 120 ataaacacta gcactttgga tttgagcggcgttacagaca aggtcaatat caacaagctc 180 attacagctt ccactaatgt gaacattaaaaactttgaca ttaaggaatt ggtggttaca 240 acccgtgttc aaagttttgg gcaatacactatttttggcg aaaatatagg cgataagtct 300 cgcattggtg tcgttagttt gcaaacgggatatagcccgg cctattctgg gggcgttact 360 tt 362 229 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 229 gtggatgctcatacggctaa ctttaatggc aatatttatc tgggaaaatc cacgaattta 60 agagtgaatggccatagcgc tcatttttaa aatattgatg ccagcaagag cgataacggg 120 ctaaacactagcaccttgga tttcagtggc gttacagaca aagtcaatat caacaagctc 180 actacatctgccactaatgt gaacgttaaa aactttgata ttaaggaatt ggtggttaca 240 acccgagttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtcg 300 cgcattggtgtcgtgagttt gcaaacggga tatagcccag cttattctgg gggcgttact 360 tt 362 230362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 230 gtggatgctc atacggctaa ctttaatggc aatatttatt tgggaaaatccacgaatttg 60 agagtgaatg gccatagcgc tcattttaaa aatattgatg ccagtaagagcgataacggg 120 ctaaacacta gcgctttgga ttttagcggc gttacagaca aagttaatatcaacaagctc 180 actacatctg ccactaatgt gaacgttaaa aactttgaca ttaaggaattggtggttaca 240 acccgagttc aaagttttgg gcaatacact atttttggcg aaaatataggcgataagtct 300 cgcattggtg tcgtgagttt gcaaacggga tatagccctg cttattctgggggcgttact 360 tt 362 231 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 231 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 agagtgaatg gccatagcgc tcattttaaaaatattgatg ccagtaagag cgataacggg 120 ctaaacacta ccactttgga tttcagcggcgttacagata aagtcaatat caacaagctc 180 actacatctg ccactaatgt gaacattaaaaactttgaca ttaaggaatt agtggttaca 240 acccgagttc agagttttgg gcaatacactatttttggcg aaaatatagg cgataagctg 300 cacattggtg tcgtgagttt gcaaacgggatatagcccag cctattctgg ggggcttact 360 tt 362 232 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 232 gtggatgctcatacagctta ttttaatggc aatatttatc tgggaaaatc cacgaattta 60 aaagtgaatggccatagcgc tcattttaaa aatattgatg ctagtaagag cgataacggg 120 ctaaacactagcgctttgga tttgagcggc gttacaaaca aggtcaatat caacaagctc 180 actacagctgccactaatgt gaacattaaa aactttgaca ttaaggaatt ggtggttaca 240 acccgcgttcagagttttgg gcaatacact atttttggcg aaaatatagg cgataagtcg 300 cgcattggtgtcgttagttt gcaaactggc tatagcccgg cctattctgg gggcgttact 360 tt 362 233362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 233 atggatgctc atacagctta ttttaatggc aatatttatc tgggaaaatccacgaattta 60 aaagtgaatg gccatagcgc tcattttaaa aatattgatg ccactaagagcgataatggt 120 ctaaacacta gcgctttgga tttgagcggc gttacaaaca aggtcaatatcaacaagctc 180 actacagctg ccactaatgt gtccattaaa aactttgaca ttaaggaattagtggttacg 240 acccgtgttc agagttttgg gcaatacact atttttggcg aaaatataggcgatcaatcg 300 cgcattggtg tcgttagttt gcaaactggc tatagcccgg cctattctgggggcgttact 360 tt 362 234 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 234 gtggatgctc atacagctta ttttaatggcaatatttatc tgggaaaatc cacgaattta 60 aaagtgaatg gccatagcgc tcattttaaaaatattgatg ccactaagag cgataatggt 120 ctatacacta gcgctttgga tttgagcggcgttacaaaca aggtcaatat taacacgctc 180 actacagctg ccactaatgt gtccattaaaaactttgaca ttaaggaatt agtggttacg 240 acccgtgttc agagttttgg gcaatacactatttttggcg aaaatatagg cgatcaatcg 300 cgcattggtg tcgttagttt gcaaactggctatagcccgg cctattctgg gggcgttact 360 tt 362 235 362 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 235 gtggatgcccatacgatcaa ttttaatggc aatatgtatt tgggaagatt tacgcattta 60 aaagtgaatggtcatacagc caattttaaa gatattgatg ccagcaaggg tagaaatggt 120 atcgacaccaccattttgga ttttagcggc gttacaaaca aggtcaatat caacaagctc 180 accacagctgccactaatgc ggccattaaa aattttgaca ttaaggaatt ggttgttaca 240 accaatgttttgagtgtggg gaaatacact gattttaccg aagatatagg cgatcaatcc 300 cgcattggtatcgtgcgttt gcaaatggga tatagcccgg cctattctgg gggcgttact 360 tt 362 236362 DNA Artificial Sequence Helicobacter pylori vacA nucleic acidsequence 236 gtggatgccc atacgatcaa ttttaatggc aatatgtatt tgggaagattcacgcattta 60 aaagtgaatg gtcatacagc caattttaaa gatattgatg ccagcaagggtagaaatggt 120 atcgacacca ccattttgga ttttagcggc gttacaaaca aggtcaatatcaacaagctc 180 accacagctg ccactaatgc ggccattaaa aattttgaca ttaaggaattggttgttaca 240 accaatgttt tgagtgtggg gaaatacact gattttaccg aagatataggcgatcaatcc 300 cgcattggta tcgttagttt gcaaacggga tatagcccgg cctattctgggggcgttact 360 tt 362 237 362 DNA Artificial Sequence Helicobacterpylori vacA nucleic acid sequence 237 gtggatgccc atacgatcaa ttttaatggcaacatgtatt tgggaagatt cacgcattta 60 aaagtgaatg gccatacagc caattttaaagatattgatg ccagcaaggg tagaaatggt 120 atcgacacca ctattttgga ttttagcggcgttacagaca aagtcaatat caacaagctc 180 actacagctg ccactaatgt gtccattaaaaactttgaca ttaaggaatt ggttgttaca 240 accaatgttt tgagtgtggg gaaatacactgattttaccg aagatatagg cgatcaatcg 300 cacattggtg tcgttagttt gcaaactggctatagcccgg tctattctgg gggcgttact 360 tt 362 238 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 238 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcattac ggcttccact 120 aatgtggccgctaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 239 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 239 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggtgtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg ttaaattt 288 240 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 240 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tgggataagt 180 gtgggggaatacactcattt tagcgaagat ataggaagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag atcaatcttt tctgggggtg ttaaattt 288 241 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 241 gtggatgctcatacagctaa ttttaaaggt attgatacgg gcaatggtgg tttcaacacc 60 ttagattttagtggcgttac agacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattgttgg ttaagaccaa tggggtgagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 242 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 242 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggcgttac agacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattggttg ttaagaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctgggggtg ttaaattt 288 243 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 243 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcatcac ggcttccact 120 aatgtggccgttaaaaacaa caacattaat gaattggtgg ttaaaaccaa tgggataagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaacaggcactag gtcaatcttt tctgggggtg tcaaattt 288 244 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 244 gtggatgctcatacagctaa ttttaaaggt attgatactg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaagtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaagattaa tggggtgagt 180 gtgggggaatacacttattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 245 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 245 gtggatgctcatacagctaa ttttaaaggt attgatactg gtaatggtgg tttcaacacc 60 ttagatttcagtggtgttac agacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattggtgg ttaaaaccaa tggtataagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 246 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 246 gtggatgctcatacagctaa ttttaaaggt attgatactg gtaatggtgg tttcaacacc 60 ttagatttcagtggtgttac agacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattggtgg ttaaaaccaa tggtataagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 247 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 247 gtggatgcccatacagctaa ttttaaaggt attgatactg gtaatggtgg tttcaacacc 60 ttagatttcagtggcgttac aaacaaagtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattggtgg ttaaaaccaa tggtataagc 180 gtgggggaatacactaattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 248 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 248 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 249 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 249 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgctaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 250 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 250 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggtgtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 251 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 251 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatattaaca agctcattac ggcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg ttaaattt 288 252 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 252 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgtgac aggtatagtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 253 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 253 gtggatggtcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcataac ggcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggtgtgagt 180 gtgggggaatacacttattt tagcgaagat ataggcagtc aatcgcacat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 254 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 254 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcattac ggcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggtgtgagt 180 gtgggggaatacacttattt tagcgaagat ataggcagtc aatcgcacat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 255 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 255 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac agacaaagtc aatatcaaca agctcatcac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcaccag gtcaatcttt tctgggggtg tcaaattt 288 256 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 256 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac agacaaagtc aatatcaaca agctcattac ggcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 257 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 257 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac agacaaagtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattggttg ttaaaaccaa tggggtaagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcacat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 258 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 258 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac ggcttccact 120 aatgtggccattaaaaactt caacattaat gaattgattg ttaaaaccaa tgggatgagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 259 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 259 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 260 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 260 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac ggcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgattg ttaaaaccaa tggggtgagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 261 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 261 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttggattttagtggcgttac agacaaagtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattgttgg ttaagaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttagaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 262 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 262 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcattac ggcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaagaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 263 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 263 gtggatgctcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aggtaaggtc aatatcaaca agctcattac ggcttccact 120 aatgtagccgttaaaaactt caacattaat gaattgttgg ttaagaccaa tggggtgagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 264 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 264 gtggatggtcatacagctaa ttttaaaggt attgatacgg gtaatggtgg tttccacacc 60 ttagattttagtggtgttac aggtaaggtc catatccaca agctcattac ggcttccact 120 aatgtggccgttaaaaactt ccacattaat gaattgattg gtaaaaccaa tgggataagt 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatcttt tctgggggtg tcaaattt 288 265 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 265 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttggaaactggcaccag gtcaatctat tttgggggtg ttaaatta 288 266 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 266 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagactttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctgggggtg ttaaattt 288 267 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 267 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagactttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcacat caacaccgtg 240 cgtttagaaactggcactag gtcaatctat tctgggggtg ttaagttt 288 268 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 268 gcgagacgtcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattagt gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcggagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctgggggtg ttaagttt 288 269 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 269 gtggatgcccatacggtcaa ttttaaaggt attgatactg gtaatggtgg tttcaacacc 60 ttagatttcagtggtgttac agacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattggtgg ttaaaaccaa tggtataagc 180 gtgggggaatacactcattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttggaaactggcactag gtcaatctat tctggcggtg ttaaattt 288 270 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 270 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagactttagtggtgttac aaacaaggtc aatatcaaca aactcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttagaaactggcaccag gtcaatctat tctgggggtg ttaagttt 288 271 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 271 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagactttagtggtgttac aaacaaggtc aatatcaaca aactcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcagtc aatcgcgcat caataccgtg 240 cgtttagaaactggcaccag gtcaatctat tctgggggtg ttaagttt 288 272 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 272 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caataccgtg 240 cgtttagaaactggcactag gtcaatctat tctgggggtg ttaagttt 288 273 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 273 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagattttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttggaaactggcactag gtcaatctat tctgggggtg ttaagttt 288 274 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 274 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagactttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccgttaaaaactt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttagaaactggcactag gtcaatctat tctgggggtg ttaaattt 288 275 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 275 gcgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaatacc 60 ttagactttagtggtgttac aaacaaggtc aatatcaaca agctcattac agcttccact 120 aatgtggccattaaaaattt caacattaat gaattgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcagtc aatcgcgcat caacaccgtg 240 cgtttagaaactggcactag gtcaatctat tctgggggtg ttaagttt 288 276 288 DNA ArtificialSequence Helicobacter pylori vacA nucleic acid sequence 276 acgagcgctcatacggtcaa ttttaaagat attgatactg gtaatggtgg tttcaacacc 60 ttagactttagtggtgttac aaacaaggtc aacatcaaca agctcattac agcttccact 120 aatgtggccattaaaaactt caacattaat gagttgttgg ttaaaaccaa tgggataagt 180 gtgggggaatacactaattt tagcgaagat ataggcaatc aatcgcgcat caacaccgtg 240 cgtttagaaactggcactag gtcaatctat tctgggggtg ttaagttt 288 277 21 DNA ArtificialSequence vacA primer 277 atggaaatac aacaaacaca c 21 278 19 DNAArtificial Sequence vacA primer 278 ctgcttgaat gcgccaaac 19 279 21 DNAArtificial Sequence 279 cacagccact ttcaataacg a 21 280 20 DNA ArtificialSequence vacA primer 280 cgtcaaaata attccaaggg 20

What is claimed is:
 1. A method for the detection of Helicobacter pylori(H. pylori) present in a sample comprising the steps of: amplifying thepolynucleic acids of the m and s regions of the vacA gene with a pair ofprimers, wherein one of said primers is selected from the groupconsisting of: SEQ ID NOS: 14-18 and, another of said primers isselected from the group consisting of SEQ ID NOS: 23-26 and 277;hybridizing the polynucleic acids obtained with at least one probehybridizing to a conserved region of the vacA gene and at least oneprobe hybridizing to a variable region of the vacA gene, thus forminghybrids; detecting the hybrids formed; and determining the presence orabsence of H. pylori in a sample from the hybridization signalsobtained.
 2. A method according to claim 1 wherein said primer pair forthe amplification step comprises VA1F (SEQ ID NO:277) and VA1XR (SEQ IDNO:14).
 3. A method according to claim 1, wherein the hybridization stepcomprises hybridizing the polynucleic acids obtained in theamplification step with a set of probes, under hybridization and washconditions, comprises at least one probe hybridizing to a conservedregion of the vacA gene of H. pylori, and at least one vacA-derivedprobe selected from the group consisting of SEQ ID NOS:35-39.
 4. A probefor use in a method of detecting the presence of H. pylori, said probecomprising a sequence selected from the group consisting of SEQ IDNOS:35-39.
 5. The method of claim 1 additionally comprising the step ofreleasing, isolating, or concentrating the H. pylori polynucleic acidsin the original sample.
 6. The method according to claim 1, wherein thehybridization step is a reverse hybridization step, wherein the probesare immobilized on a solid support.
 7. The method according to claim 6,wherein said probes are immobilized as parallel lines on a solidsupport.
 8. The method according to claim 6, wherein said solid supportis a membrane strip.
 9. A kit for detecting and/or typing H. pyloristrains in a sample liable to contain it, comprising the followingcomponents: at least one probe selected from the group consisting of:SEQ ID NOS:1-11 and 27-39 or variants thereof, with said probe and/orother probes applied; a buffer or components necessary to produce thebuffer enabling an amplification or a hybridization reaction betweensaid probes and the amplified products; and a means for detecting thehybrids resulting from said hybridization.
 10. The method according toclaim 9, wherein said solid support is a microtiter plate.
 11. Themethod of claim 1, further comprising amplifying the polynucleic acidsof the cagA gene of H. pylori with a primer pair that amplifies aconserved region of the cagA gene of all H.pylori strains.
 12. Themethod of claim 5, wherein each primer from said primer pair comprises aprimer selected from the group consisting of: SEQ ID NOS: 1, 12-13,19-22, and
 27. 13. A method according to claim 1 for the detectionand/or typing of alleles of the cagA and vacA gene of H.pylori presentin a sample using a set of probes and/or primers specially designed todetect and/or to amplify and/or to type the said alleles, with saidprobes selected from the group consisting of: SEQ ID NOS: 1-11 and 27-39and primers being selected from the group consisting of: SEQ ID NOS:12-26 and variants thereof that can amplify said vacA or cagA region ofall strains of H.pylori.
 14. An isolated vacA polynucleotide sequenceselected from the group consisting of: SEQ ID NOS: 40-91 and SEQ ID NOS:115-276.
 15. A method for the detection and/or typing of Helicobacterpylori (H.pylori) strains present in a sample comprising the steps of:amplifying the polynucleic acids of the m and s regions of the vacA geneand a conserved region of the cagA gene, with a pair of primers, whereinsaid vacA primers are selected from the group consisting of: SEQ ID NOS:14-18, 23-26, and 277; hybridizing the polynucleic acids obtained withat least one probe hybridizing to a conserved region of the cagA geneand at least one probe hybridizing to a variable region of the vacAgene, thus forming hybrids; detecting the hybrids formed; detectingand/or typing H.pylori strains present in a sample from the differentialhybridization signals obtained; wherein said typing comprises theallele-specific detection of a strain according to the vacA polynucleicacid alleles.
 16. The method of claim 11, wherein said cagA primers areselected from the group consisting of SEQ ID NOS:12-13, and 19-22. 17.The method of claim 1, wherein said probes has compatible hybridizationand wash conditions.
 18. The method of claim 11 wherein thehybridization step is a reverse hybridization step, wherein said probesare immobilized on a solid support.
 19. The method according to claim 11wherein the polynucleic acids obtained in the amplification step areimmobilized on a solid support and the subsequent hybridization step iscarried out on said solid support.
 20. A probe for use in a methodaccording to claim 11, wherein said vacA probe is selected from thegroup consisting of: SEQ ID NOS: 2-11 and 28-34.
 21. An oligonucleotideprimer, wherein said vacA primer is selected from the group consistingof SEQ ID NOS: 14-18, and 23-26.
 22. A probe for detection or typing ofvacA, said probe selected from the group consisting of SEQ ID NOS:2-11,and 28-39.
 23. A vacA-specific oligonucleotide selected from the groupconsisting of SEQ ID NOS: 14-18 and 23-26.
 24. A method according toclaim 11 wherein at least one of said probes is selected from the groupconsisting of SEQ ID NOS:2-11 and 28-39 and wherein said primers areselected from the group consisting of SEQ ID NOS:14-18 and 23-26.